Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca²⁺-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca²⁺-dependent manner (with optimal 61 μm free Ca²⁺ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca²⁺, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (K_d = 1.7 × 10^–5 m) than for the C2F interaction (K_d = 2.6 × 10^–9 m). In contrast, it was the otoferlin C2D domain that bound the Ca_v1.3 II-III loop, in a Ca²⁺-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca²⁺ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Ca_v1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref. 1). In mechanosensory hair cells, calcium microdomains (2) and possibly nanodomains (3) are formed when voltage-gated calcium channels open upon depolarization. Calcium at these sites is thought to activate protein interactions, leading to vesicle fusion. Some of the key players in this process are the target-SNARE² proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin (4). Vesicle-SNARE synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal synapse, modulating calcium channels and vesicle release by a Ca²⁺-dependent interaction with other SNARE proteins in the presence of lipid molecules (4–6). However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not detected (7). Instead, fast neurotransmitter release in auditory and vestibular hair cells, facilitated largely by an L-type voltagegated calcium channel, Ca_v1.3 (8, 9), is thought to be modulated by a newly discovered protein, otoferlin, acting as the Ca²⁺ sensor and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic deafness (10). Gene sequences of different deaf families show that the OTOF gene can undergo mutation at multiple locations (11–13). Recently, it has been demonstrated that otoferlin is necessary for synaptic exocytosis from hair cells (14). Further, an engineered mutation in the C2B domain of otoferlin has been shown to cause deafness in mice (15). However, the precise function of otoferlin as a synaptic protein is not well understood.Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in humans have been documented to cause DFNB9 deafness (11, 12). Previous studies suggested that a region of otoferlin containing all three C2 domains, D, E, and F, binds directly to the t-SNARE molecules syntaxin 1A and SNAP-25 in response to an increase in Ca²⁺ concentration (14). However, it is not understood how a single amino acid substitution in one domain of otoferlin, such as C2F (11) or C2D (12), might independently lead to deafness. Here, we examine the role of otoferlin as a Ca²⁺ sensor as well as a facilitator of vesicle fusion, as indicated by protein-protein interactions and their [Ca²⁺] dependence.     

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic ��-Cells

Ryanodine receptors (RyR) are Ca²⁺ channels that mediate Ca²⁺ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca²⁺ entry that was independent of store-operated Ca²⁺ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ_K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP₃ receptors (IP₃R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP₃R3, we showed that RyR2 mediates both the Ca²⁺ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca²⁺ entry.Ryanodine receptors (RyR)³ and inositol 1,4,5-trisphosphate receptors (IP₃R) (1, 2) are the archetypal intracellular Ca²⁺ channels. Both are widely expressed, although RyR are more restricted in their expression than IP₃R (3, 4). In common with many cells, pancreatic β-cells and insulin-secreting cell lines express both IP₃R (predominantly IP₃R3) (5, 6) and RyR (predominantly RyR2) (7). Both RyR and IP₃R are expressed mostly within membranes of the endoplasmic (ER), where they mediate release of Ca²⁺. Functional RyR are also expressed in the secretory vesicles (8, 9) or, and perhaps more likely, in the endosomes of β-cells (10). Despite earlier suggestions (11), IP₃R are probably not present in the secretory vesicles of β-cells (8, 12, 13).All three subtypes of IP₃R are stimulated by IP₃ with Ca²⁺ (1), and the three subtypes of RyR are each directly regulated by Ca²⁺. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca²⁺ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca²⁺ release and insulin secretion in β-cells (8, 14). The activities of both families of intracellular Ca²⁺ channels are also modulated by many additional signals that act directly or via phosphorylation (15, 16). Although they commonly mediate release of Ca²⁺ from the ER, both IP₃R and RyR select rather poorly between Ca²⁺ and other cations (permeability ratio, P_Ca/P_K ∼7) (1, 17). This may allow electrogenic Ca²⁺ release from the ER to be rapidly compensated by uptake of K⁺ (18), and where RyR or IP₃R are expressed in other membranes it may allow them to affect membrane potential.Both Ca²⁺ entry and release of Ca²⁺ from intracellular stores contribute to the oscillatory increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (7). Glucose rapidly equilibrates across the plasma membrane (PM) of β-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K_ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca²⁺ channels, predominantly L-type Ca²⁺ channels (21). The resulting Ca²⁺ entry is amplified by Ca²⁺-induced Ca²⁺ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close K_ATP channels, in the treatment of type 2 diabetes. Ca²⁺ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (23). However, neither the increase in [Ca²⁺]_i nor the insulin release evoked by glucose or other nutrients is entirely dependent on Ca²⁺ entry (7, 24) or closure of K_ATP channels (25). This suggests that glucose metabolism may also more directly activate RyR (7, 26) and/or IP₃R (27) to cause release of Ca²⁺ from intracellular stores. A change in the ATP/ADP ratio is one means whereby nutrient metabolism may be linked to opening of intracellular Ca²⁺ channels because both RyR (28) and IP₃R (1) are stimulated by ATP.The other major physiological regulators of insulin release are the incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic hormone (29). These hormones, released by cells in the small intestine, stimulate synthesis of cAMP in β-cells and thereby potentiate glucose-evoked insulin release (30). These pathways are also targets of drugs used successfully to treat type 2 diabetes (29). The responses of β-cells to cAMP involve both cAMP-dependent protein kinase and epacs (exchange factors activated by cAMP) (31, 32). The effects of the latter are, at least partly, due to release of Ca²⁺ from intracellular stores via RyR (33–35) and perhaps also via IP₃R (36). The interplays between Ca²⁺ and cAMP signaling generate oscillatory changes in the concentrations of both messengers (37). RyR and IP₃R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.     

Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain

The Ca²⁺ release-activated Ca²⁺ channel is a principal regulator of intracellular Ca²⁺ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca²⁺ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca²⁺ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150Å in height and 95Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.Ca²⁺ is an intracellular second messenger that plays important roles in various physiological functions such as immune response, muscle contraction, neurotransmitter release, and cell proliferation. Intracellular Ca²⁺ is mainly stored in the endoplasmic reticulum (ER).² This ER system is distributed through the cytoplasm from around the nucleus to the cell periphery close to the plasma membrane. In non-excitable cells, the ER releases Ca²⁺ through the inositol 1,4,5-trisphosphate (IP₃) receptor channel in response to various signals, and the Ca²⁺ store is depleted. Depletion of Ca²⁺ then induces Ca²⁺ influx from outside the cell to help in refilling the Ca²⁺ stores and to continue Ca²⁺ rise for several minutes in the cytoplasm (1, 2). This Ca²⁺ influx was first proposed by Putney (3) and was named store-operated Ca²⁺ influx. In the immune system, store-operated Ca²⁺ influx is mainly mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) current, which is a highly Ca²⁺-selective inwardly rectified current with low conductance (4, 5). Pathologically, the loss of CRAC current in T cells causes severe combined immunodeficiency (6) where many Ca²⁺ signal-dependent gene expressions, including cytokines, are interrupted (7). Therefore, CRAC current is necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically characterized as essential components of the CRAC channel (8–12). They are separately located in the plasma membrane and in the ER membrane; co-expression of these proteins presents heterologous CRAC-like currents in various types of cells (10, 13–15). Both of them are shown to be expressed ubiquitously in various tissues (16–18). STIM1 senses Ca²⁺ depletion in the ER through its EF hand motif (19) and transmits a signal to Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory component for some transient receptor potential canonical channels (20, 21), it is believed from the mutation analyses to be the pore-forming subunit of the CRAC channel (8, 22–24). In the steady state, both Orai1 and STIM1 molecules are dispersed in each membrane. When store depletion occurs, STIM1 proteins gather into clusters to form puncta in the ER membrane near the plasma membrane (11, 19). These clusters then trigger the clustering of Orai1 in the plasma membrane sites opposite the puncta (25, 26), and CRAC channels are activated (27).Orai1 has two homologous genes, Orai2 and Orai3 (8). They form the Orai family and have in common the four transmembrane (TM) segments with relatively large N and C termini. These termini are demonstrated to be in the cytoplasm, because both N- and C-terminally introduced tags are immunologically detected only in the membrane-permeabilized cells (8, 9). The subunit stoichiometry of Orai1 is as yet controversial: it is believed to be an oligomer, presumably a dimer or tetramer even in the steady state (16, 28–30).Despite the accumulation of biochemical and electrophysiological data, structural information about Orai1 is limited due to difficulties in purification and crystallization. In this study, we have purified Orai1 in its tetrameric form and have reconstructed the three-dimensional structure from negatively stained electron microscopic (EM) images.     

Specific Role of Neuronal Nitric-oxide Synthase when Tethered to the Plasma Membrane Calcium Pump in Regulating the ��-Adrenergic Signal in the Myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates β-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser¹⁶ phospholamban (PLB) phosphorylation as well as Ser²² and Ser²³ cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the β-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.Neuronal nitric-oxide synthase (nNOS)⁵ is involved in a number of key processes in cardiomyocytes including calcium cycling (1), the β-adrenergic contractile response (2, 3), post-infarct left ventricular remodeling (4), and the regulation of redox equilibrium (5). Moreover, a polymorphism in an nNOS-interacting protein, CAPON, has been found to form a quantitative trait for the determination of the QT interval in humans (6), whereas a mutation in α1-syntrophin (SNTA1), another interacting partner of nNOS, has been associated with long QT syndrome (7). The signaling events downstream of the nNOS-CAPON (8) and nNOS-SNTA1 (7) complexes, which are responsible for mediating cardiac repolarization and sodium current respectively, have been elucidated. The nNOS-containing protein complex is therefore of immediate relevance to human pathology.In recent years, we have shown that the sarcolemmal calcium pump, which ejects calcium to the extracellular compartment (reviewed in Refs. 9 and 10), is an important molecule involved in signal regulation and transmission in the heart (11). We have demonstrated that isoform 4b of the sarcolemmal calcium pump (also known as PMCA4b for plasma membrane calcium/calmodulin-dependent ATPase 4b) modulates signaling through a tight molecular interaction with nNOS, leading to the modulation of β-adrenergic responsiveness in the heart (12). However, the events following signaling through the PMCA4b-nNOS complex remain unknown.In myocardial cells, nNOS has been localized to the sarcolemma (13), sarcoplasmic reticulum (2), and mitochondria (14), and translocation between compartments has been demonstrated (15). It has been speculated that these various localizations provide specificity to NO signaling, but the exact mechanisms have yet to be elucidated. In this study, we show a mechanism by which one fraction of nNOS serves highly specific functions through binding to PMCA4b. As PMCA4b is confined to the sarcolemma and is a calcium pump, it is the first identified protein to fulfill these aggregate functions. 1) It acts as an anchoring protein; 2) it regulates nNOS activity; and 3) it modulates a process at the plasma membrane, i.e. β-adrenergic signaling.     

Distinct Mechanisms of Regulation by Ca2+/Calmodulin of Type 1 and 8 Adenylyl Cyclases Support Their Different Physiological Roles

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca²⁺/calmodulin (CaM), have discrete neuronal functions. Although the Ca²⁺ sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca²⁺/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca²⁺/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca²⁺/CaM both in vivo and in vitro and explore the different role of each Ca²⁺-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca²⁺ entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca²⁺ events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca²⁺ dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.Nine membrane-bound mammalian adenylyl cyclases (ACs),² AC1–AC9, have been identified (1). They possess a common predicted structure (2)³ and are stimulated by forskolin (FSK; except AC9) and G_sα, although they are distributed and regulated differently (1, 3, 4). Four ACs are regulated by physiological concentrations of Ca²⁺ and thereby provide a critical link between the Ca²⁺- and cAMP-signaling pathways (3, 5); AC5 and AC6 are directly inhibited by Ca²⁺, whereas AC1 and AC8 are stimulated by Ca²⁺ in a calmodulin (CaM)-dependent manner (5). AC3 is also regulated by CaM in vitro, although this requires supramicromolar concentration of Ca²⁺ (6), and in vivo AC3 is inhibited by Ca²⁺ via CaM kinase II (7), unlike AC1 and AC8.AC1 is closely related in sequence to the Ca²⁺/CaM-stimulable rutabaga AC from Drosophila, which is important in Drosophila learning tasks (8–10). AC1 and the other Ca²⁺/CaM-stimulable mammalian AC, AC8, have also been implicated in learning and memory (11). As a means of establishing their proposed roles, single and/or double AC1 and AC8 knockout mice have been generated. Mouse models have demonstrated that Ca²⁺/CaM-stimulable ACs are involved in long-term potentiation and long-term memory (12). However, despite the general view that AC1 and AC8 can behave similarly, discrete physiological actions of each isoform are becoming apparent. Recent studies by Zhuo''s group demonstrated that AC1 specifically participates in N-methyl-d-aspartic acid receptor-induced neuronal excitotoxicity (13) and an increase in GluR1 synthesis induced by blocking AMPA receptors (14). Furthermore, Nicol and colleagues (15, 16) showed a contribution of AC1, but not AC8, in axon terminal refinement in the retina. On the other hand, AC8 specifically was seen to be responsible for retrieval from adaptive presynaptic silencing (17) and the acquiring of new spatial information (18). These differences in physiological roles must reflect not only differences in their distributions but also presumably in their regulatory properties. Both enzymes are expressed in brain; AC1 is neuro-specific, whereas the expression of AC8 is more widespread (1, 12). Within the central nervous system, AC1 is abundant in the hippocampus, the cerebral cortex, and the granule cells of the cerebellum, whereas AC8 has a high expression level in the thalamus and the cerebral cortex (19). Studies of mouse brain revealed that AC1 is distributed pre-synaptically and AC8 post-synaptically (18, 20).Although physiological differences in the roles of these two enzymes are suggested from the studies outlined above, the regulatory mechanisms that might underlie these differences are not. AC1 is more sensitive to Ca²⁺ than is AC8 in vitro (21, 22), yet details on how these two enzymes are regulated by Ca²⁺/CaM are sparse. In non-excitable cells, a Ca²⁺ elevation caused by capacitative Ca²⁺ entry (CCE), the mode of Ca²⁺ entry triggered by emptying Ca²⁺ from internal stores (23), preferentially stimulates AC1 and AC8 (21). Although stimulation of AC8 by CCE has been shown to be at least partially dependent on its localization at lipid rafts (24), whether AC1 is also targeted to this region of plasma membranes has never been addressed. In addition, CaM regulation of AC1 and AC8 has not been compared in detail, although CaM appears to bind to different domains of the two enzymes. AC8 utilizes two CaM binding domains: a classic amphipathic “1-5-8-14” motif at the N terminus and an IQ-like motif in the C2b domain (25). A recent study indicates that CaM pre-associates with the N terminus of AC8, where it becomes fully saturated upon a Ca²⁺ rise, and activates the enzyme via a C-terminally mediated relief of auto-inhibitory mechanisms (26). By contrast, only residues 495–522 of the C1b region of AC1 have been shown to bind CaM in a Ca²⁺-dependent manner (27, 28). With the presence of only one CaM binding domain in AC1, a simpler mechanism of CaM regulation might be expected.CaM mediates the regulation of numerous Ca²⁺-dependent processes in eukaryotic cells (29). The protein possesses N- and C-terminal lobes, both of which contain two Ca²⁺ binding EF hands (EF1 and EF2 in the N lobe, and EF3 and EF4 in the C lobe (30)). Mutations in the EF hands have been valuable for investigating the interaction of CaM with its targets. Alanine substitutions in the EF12 pair or EF34 pair have generated CaM₁₂ and CaM₃₄ to investigate the independent function of the C and N lobes of CaM, respectively (31, 32).Against the background of the distinct physiological roles carried out by AC1 and AC8, we performed a detailed comparison of the two enzymes expressed in HEK 293 cells. Their sensitivity to Ca²⁺/CaM was compared both in vitro and in vivo; the possibility that they might be expressed in different domains of the plasma membrane was addressed; and putative lobe-specific CaM regulation was assessed using Ca²⁺-binding mutants of CaM. Single cell measurements using a FRET-based cAMP sensor were performed to compare the kinetic responses of the enzymes to physiological elevations of [Ca²⁺]_i. The results demonstrate superficial similarities in the regulation of AC1 and AC8 but critical disparities in their mechanism of activation by the lobes of CaM and in the speed and pattern of their responsiveness to [Ca²⁺]_i. These discrete behaviors provide a physiological opportunity for different outcomes to elevation of [Ca²⁺]_i, depending on the AC that is expressed in particular contexts.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca²⁺-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Here we present NMR structures of CaBP1 in both Mg²⁺-bound and Ca²⁺-bound states and their structural interaction with InsP₃Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg²⁺ bound at EF1. The C-domain binds Ca²⁺ at EF3 and EF4, and exhibits a Ca²⁺-induced closed to open transition like that of CaM. The Ca²⁺-bound C-domain contains exposed hydrophobic residues (Leu¹³², His¹³⁴, Ile¹⁴¹, Ile¹⁴⁴, and Val¹⁴⁸) that may account for selective binding to InsP₃Rs. Isothermal titration calorimetry analysis reveals a Ca²⁺-induced binding of the CaBP1 C-domain to the N-terminal region of InsP₃R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP₃Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP₃ binding to the receptor. We propose that CaBP1 may regulate Ca²⁺-dependent activity of InsP₃Rs by promoting structural contacts between the suppressor and core domains.Calcium ion (Ca²⁺) in the cell functions as an important messenger that controls neurotransmitter release, gene expression, muscle contraction, apoptosis, and disease processes (1). Receptor stimulation in neurons promotes large increases in intracellular Ca²⁺ levels controlled by Ca²⁺ release from intracellular stores through InsP₃Rs (2). The neuronal type-1 receptor (InsP₃R1)² is positively and negatively regulated by cytosolic Ca²⁺ (3-6), important for the generation of repetitive Ca²⁺ transients known as Ca²⁺ spikes and waves (1). Ca²⁺-dependent activation of InsP₃R1 contributes to the fast rising phase of Ca²⁺ signaling known as Ca²⁺-induced Ca²⁺ release (7). Ca²⁺-induced inhibition of InsP₃R1, triggered at higher cytosolic Ca²⁺ levels, coordinates the temporal decay of Ca²⁺ transients (6). The mechanism of Ca²⁺-dependent regulation of InsP₃Rs is complex (8, 9), and involves direct Ca²⁺ binding sites (5, 10) as well as remote sensing by extrinsic Ca²⁺-binding proteins such as CaM (11, 12), CaBP1 (13, 14), CIB1 (15), and NCS-1 (16).Neuronal Ca²⁺-binding proteins (CaBP1-5 (17)) represent a new sub-branch of the CaM superfamily (18) that regulate various Ca²⁺ channel targets. Multiple splice variants and isoforms of CaBPs are localized in different neuronal cell types (19-21) and perform specialized roles in signal transduction. CaBP1, also termed caldendrin (22), has been shown to modulate the Ca²⁺-sensitive activity of InsP₃Rs (13, 14). CaBP1 also regulates P/Q-type voltage-gated Ca²⁺ channels (23), L-type channels (24), and the transient receptor potential channel, TRPC5 (25). CaBP4 regulates Ca²⁺-dependent inhibition of L-type channels in the retina and may be genetically linked to retinal degeneration (26). Thus, the CaBP proteins are receiving increased attention as a family of Ca²⁺ sensors that control a variety of Ca²⁺ channel targets implicated in neuronal degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in CaM and troponin C (18) (Fig. 1). By analogy to CaM (27), the four EF-hands are grouped into two domains connected by a central linker that is four residues longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain non-conserved amino acids within the N-terminal region that may confer target specificity. Another distinguishing property of CaBPs is that the second EF-hand lacks critical residues required for high affinity Ca²⁺ binding (17). CaBP1 binds Ca²⁺ only at EF3 and EF4, whereas it binds Mg²⁺ at EF1 that may serve a functional role (28). Indeed, changes in cytosolic Mg²⁺ levels have been detected in cortical neurons after treatment with neurotransmitter (29). Other neuronal Ca²⁺-binding proteins such as DREAM (30), CIB1 (31), and NCS-1 (32) also bind Mg²⁺ and exhibit Mg²⁺-induced physiological effects. Mg²⁺ binding in each of these proteins helps stabilize their Ca²⁺-free state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary structural elements (α-helices and β-strands) were derived from NMR analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted green, red, cyan, and yellow. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Non-conserved residues in the hydrophobic patch are colored red.Despite extensive studies on CaBP1, little is known about its structure and target binding properties, and regulation of InsP₃Rs by CaBP1 is somewhat controversial and not well understood. Here, we present the NMR solution structures of both Mg²⁺-bound and Ca²⁺-bound conformational states of CaBP1 and their structural interactions with InsP₃R1. These CaBP1 structures reveal important Ca²⁺-induced structural changes that control its binding to InsP₃R1. Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca²⁺-induced binding to the N-terminal cytosolic region of InsP₃R1. We propose that CaBP1 may regulate Ca²⁺-dependent channel activity in InsP₃Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca²⁺-dependent channel gating (8, 33, 34).     

Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and l-citrulline from l-arginine and O₂, with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS)² in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca²⁺ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca²⁺)₄-CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca²⁺-binding sites, enfold the domain, as has been observed in several other such CaM-peptide complexes (7). Consistent with this structure, investigations of CaM-dependent activation of the neuronal synthase suggest that both CaM lobes must participate (8, 9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (10–12). There are equivalent phosphorylation sites in the human enzyme (10–12). Phosphorylation of the bovine enzyme at Thr-497, which is located in the CaM-binding domain, blocks CaM binding and enzyme activation (7, 11, 13, 14). Ser-116 can be basally phosphorylated in cells (10, 11, 13, 15), and dephosphorylation of this site has been correlated with increased NO production (13, 15). However, it has also been reported that a phosphomimetic substitution at this position has no effect on enzyme activity measured in vitro (13). Ser-1179 is phosphorylated in response to a variety of stimuli, and this has been reliably correlated with enhanced NO production in cells (10, 11). Indeed, NO production is elevated in transgenic endothelium expressing an eNOS mutant containing an S1179D substitution, but not in tissue expressing an S1179A mutant (16). Shear stress or insulin treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in endothelial cells, and this is correlated with increased NO production in the absence of extracellular Ca²⁺ (17–19). Akt-catalyzed phosphorylation or an S1179D substitution has also been correlated with increased synthase activity in cell extracts at low intracellular free [Ca²⁺] (17). Increased NO production has also been observed in cells expressing an eNOS mutant containing an S617D substitution, and physiological stimuli such as shear-stress, bradykinin, VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 (12, 13, 20). Although S617D eNOS has been reported to have the same maximum activity in vitro as the wild type enzyme (20), in our hands an S617D substitution increases the maximal CaM-dependent synthase activity of purified mutant enzyme ∼2-fold, partially disinhibits reductase activity, and reduces the EC₅₀(Ca²⁺) values for CaM binding and enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp substitution at Ser-1179 in eNOS on the Ca²⁺ dependence of CaM binding and CaM-dependent activation of reductase and synthase activities. We also describe the effects on these properties of combining this substitution with one at Ser-617. Finally, we demonstrate that Akt-catalyzed phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar functional effects. Our results suggest that phosphorylation of eNOS at Ser-617 and Ser-1179 can substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm, while single phosphorylations at these sites produce smaller activity increases, and can do so only at higher free Ca²⁺ concentrations.     

Retinol Esterification by DGAT1 Is Essential for Retinoid Homeostasis in Murine Skin

Retinoic acid (RA) is a potent signaling molecule that is essential for many biological processes, and its levels are tightly regulated by mechanisms that are only partially understood. The synthesis of RA from its precursor retinol (vitamin A) is an important regulatory mechanism. Therefore, the esterification of retinol with fatty acyl moieties to generate retinyl esters, the main storage form of retinol, may also regulate RA levels. Here we show that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1 deficiency results in elevated levels of RA in skin and cyclical hair loss; both are prevented by dietary retinol deprivation. Further, DGAT1-deficient skin exhibits enhanced sensitivity to topically administered retinol. Deletion of the enzyme specifically in the epidermis causes alopecia, indicating that the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis. These findings show that DGAT1 functions as an ARAT in the skin, where it acts to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings may have implications for human skin or hair disorders treated with agents that modulate RA signaling.Regulation of cellular proliferation and differentiation of epithelial tissues is crucial in embryonic development and in adult homeostasis. Retinoic acid (RA)² is a major regulator of these processes (1) through its ability to serve as a ligand for RA nuclear receptors (RARs) (2). Since RA is such a potent signaling molecule, its levels must be tightly controlled. Indeed, excess RA is highly teratogenic during embryonic development and may be toxic to adult tissues (3). Further, RA is used therapeutically for skin disorders, such as acne and psoriasis, and certain cancers (4), but its uses are often limited by local and systemic toxicity. Thus, understanding how RA levels are regulated has important biological and clinical relevance.The synthesis of RA from its precursor retinol, or vitamin A, is a major node in the regulation of RA levels (5). To generate RA, retinol is oxidized in two sequential reactions, catalyzed by retinol and retinal dehydrogenases (5), whose activities regulate RA homeostasis. We hypothesized that the availability of retinol for these reactions may also be regulated by the balance between retinol and retinyl esters. Indeed, the majority of retinol in the body is stored as retinyl esters, which are concentrated in cytosolic lipid droplets of cells and serve as a local source of retinol. Retinyl esters are also stored in major organs, such as liver and white adipose tissue (WAT), from which retinol can be mobilized to supply other tissues during increased demand. Thus, retinol esterification may participate in regulating the retinol pool available for RA synthesis.Retinol esterification is carried out by two distinct enzymatic activities. One is mediated by lecithin:retinol acyltransferase (LRAT), which catalyzes the covalent joining of a fatty acyl moiety from lecithin (phosphatidylcholine) to retinol that is bound to cellular retinol-binding protein (CRBP) (6, 7). LRAT activity is crucial for maintaining tissue retinol stores. LRAT-null (Lrat^-/-) mice have severe reductions in hepatic and lung retinyl ester levels (8–10), which are accompanied by testicular hypoplasia/atrophy (9) and blindness (8). Retinyl ester levels are normal in WAT and several other tissues, indicating alternative mechanisms for retinol esterification (9, 10). This esterification is probably mediated in part by acyl CoA:retinol acyltransferase (ARAT) enzymes, which use fatty acyl-CoA and unbound retinol as substrates (11). Although many tissues exhibit ARAT activity (12), attempts to purify and clone an ARAT gene were unsuccessful, and thus molecular tools to study ARAT activity have been lacking. However, the enzyme encoded by Dgat1, an acyl CoA:diacylglycerol acyltransferase (DGAT), was recently reported to catalyze the ARAT reaction in vitro (13, 14). Moreover, several tissues of Dgat1^-/- mice had reduced ARAT activity, and retinol esterification was reduced in cultured murine embryonic fibroblasts lacking DGAT1 (14). Most recently, a study of Dgat1^-/- mice demonstrated a role for the enzyme in retinol absorption in the small intestine (15). Thus, accumulating evidence indicates that the retinol esterification activity of DGAT1 is of biological, and possibly clinical, importance.In the current study, we investigated whether retinol esterification by DGAT1 is important in murine skin. Dgat1^-/- mice exhibit a pleiotropic phenotype, which includes resistance to diet-induced obesity and altered energy metabolism but also includes prominent phenotypic findings in the skin (16–19). Retinoids play key roles in skin and hair biology (20), and excess retinoids induce epidermal hyperplasia, inhibit sebocyte proliferation and differentiation, and alter hair growth (21). Notably, the skin manifestations of Dgat1^-/- mice, which include alopecia and sebaceous gland atrophy (18), resemble those of retinoid toxicity (22, 23). Thus, we hypothesized that DGAT1 functions as an ARAT in murine skin and that the absence of DGAT1 alters retinoid homeostasis. In this study, we tested this hypothesis by examining retinoid metabolism in the skin of DGAT1-deficient mice.     

Phosphorylation of the Consensus Sites of Protein Kinase A on ��1D L-type Calcium Channel

The novel α_1D L-type Ca²⁺ channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased α_1D Ca²⁺ channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the α₁ subunit of the α_1D Ca²⁺ channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the α₁ subunit of α_1D Ca²⁺ channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the α₁ subunit of α_1D Ca²⁺ channel. These novel findings provide new insights into the autonomic regulation of the α_1D Ca²⁺ channel in the heart.L-type Ca²⁺ channels are essential for the generation of normal cardiac rhythm, for induction of rhythm propagation through the atrioventricular node and for the contraction of the atrial and ventricular muscles (1–5). L-type Ca²⁺ channel is a multisubunit complex including α₁, β and α₂/δ subunits (5–7). The α₁ subunit contains the voltage sensor, the selectivity filter, the ion conduction pore, and the binding sites for all known Ca²⁺ channel blockers (6–9). While α_1C Ca²⁺ channel is expressed in the atria and ventricles of the heart (10–13), expression of α_1D Ca²⁺ channel is restricted to the sinoatrial (SA)² and atrioventricular (AV) nodes, as well as in the atria, but not in the adult ventricles (2, 3, 10).Only recently it has been realized that α_1D along with α_1C Ca²⁺ channels contribute to L-type Ca²⁺ current (I_Ca-L) and they both play important but unique roles in the physiology/pathophysiology of the heart (6–9). Compared with α_1C, α_1D L-type Ca²⁺ channel activates at a more negative voltage range and shows slower current inactivation during depolarization (14, 15). These properties may allow α_1D Ca²⁺ channel to play critical roles in SA and AV nodes function. Indeed, α_1D Ca²⁺ channel knock-out mice exhibit significant SA dysfunction and various degrees of AV block (12, 16–19).The modulation of α_1C Ca²⁺ channel by cAMP-dependent PKA phosphorylation has been extensively studied, and the C terminus of α₁ was identified as the site of the modulation (20–22). Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a membrane-permeable cAMP analog, increased α_1D Ca²⁺ channel activity using patch clamp studies (2). However, very little is known about potential PKA phosphorylation consensus motifs on the α_1D Ca²⁺ channel. We therefore hypothesized that the C terminus of the α₁ subunit of the α_1D Ca²⁺ channel mediates its modulation by cAMP-dependent PKA pathway.     

Spermine Synthase Deficiency Leads to Deafness and a Profound Sensitivity to ��-Difluoromethylornithine

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes for ornithine decarboxylase and S-adenosylmethionine decarboxylase, which are enzymes needed for the synthesis of putrescine, spermidine, and spermine, are lethal at early stages of embryonic development (1, 2). There is convincing evidence that the formation of hypusine in eIF5A, which requires spermidine as a precursor, is essential for eukaryotes (3). However, the function(s) of spermine is not so well established. Yeast mutants with inactivated spermine synthase grow at a normal rate (4). Mammalian cells in culture also grow normally in the presence of inhibitors of spermine synthase (5) or after inactivation of the spermine synthase gene (SpmS) (6–8). Inactivation of both of the genes that were originally described as encoding spermine synthases in plants leads to profound developmental defects (9–11), but recently it was discovered that one of these genes actually encodes a thermospermine synthase, and it appears that the lack of thermospermine may be responsible for these defects (12).In contrast, spermine is clearly required for normal development in mammals. The rare human Snyder-Robinson syndrome is caused by mutations in SpmS located in the X chromosome that drastically reduces the amount of spermine synthase (13, 14). This leads to mental retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry, thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X chromosomal deletion that includes SpmS and have no detectable spermine synthase activity, do survive but are only viable on the B6C3H background (15–17). This mouse strain having an X-linked dominant mutation was isolated from a female offspring of an irradiated mouse and was termed gyro (Gy)² based on a circling behavior pattern in affected males (18). Subsequent studies have shown that the Gy mice have a deletion of part of the X chromosome that inactivates both Phex, a gene that regulates phosphate metabolism, and SpmS (16, 19). The lack of SpmS causes a total absence of spermine (6, 7, 15, 16). Such Gy mice suffer from hypophosphatemia, have a greatly reduced size, sterility, and neurological abnormalities, and have a short life span (6, 16, 18). All of these changes except the hypophosphatemia are reversed when spermine synthase activity is restored (20).The original characterization of Gy mice also reported preliminary indications that these mice had hearing defects lacking the Preyer reflex (21, 22). This is of particular interest in the context of polyamine metabolism because a drug, α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine decarboxylase has been shown to cause occasional hearing loss in some patients (23–26). Although DFMO was ineffective for cancer treatment, it is an extremely promising agent for cancer chemoprevention (27, 28). When combined with sulindac, DFMO treatment produced a substantial reduction in the recurrence of colorectal adenomas in a large clinical trial (27). DFMO is a major drug for the treatment of African sleeping sickness caused by Trypanosoma brucei (29, 30). It is also used as a topically applied cream for treatment of unwanted facial hair in women (31, 32). DFMO is generally well tolerated even at high doses, but reversible hearing loss has been reported in multiple clinical trials (25, 33), and a rarer irreversible defect has also been reported (34). These side effects are not observed at lower doses of DFMO (26, 27).Ototoxicity has been demonstrated to occur in experimental animals treated with DFMO including rats (35), guinea pigs (36), gerbils (37), and mice (38). Using immunohistochemistry, a high level of ornithine decarboxylase was observed in the inner ear of the rat, with the highest in the organ of Corti and lateral wall followed by the cochlear nerve (39). Measurements of polyamines in the relevant structures are very difficult due to the small amount of tissue available, but as expected, DFMO treatment reduced polyamine levels and ornithine decarboxylase activity in the inner ear of the guinea pig (36). A plausible explanation for the importance of polyamines in auditory physiology is based on their well documented role as regulators of potassium channels (38). The inward rectification of Kir channels is caused by blockage of the outward current by polyamines (40–42). Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel showed that inward rectification was reduced and that there was a marked reduction in endocochlear potential (EP). It was proposed that DFMO treatment increases the outward Kir4.1 current, resulting in a drop in EP (38).In the experiments reported here, we have studied in more detail the role of polyamines in auditory physiology using Gy mice and crosses of these mice with transgenic CAG/SpmS mice (43). These mice express spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter, which was designed to provide ubiquitous expression (44–46). Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that there was a high level of expression of the transgene in many different organs and that this level was maintained for at least 1 year (43). Our studies confirm that Gy mice are totally deaf and that this condition is reversed by the expression of the SpmS gene. These changes are due to a virtually complete loss of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy mice. Unexpectedly, it was found that these mice show a rapid and profound toxicity to this drug, leading to death within a few days. Within 5 days of exposure to DFMO in the drinking water, the DFMO-treated mice suffered a catastrophic loss of balance due to inner ear effects. This toxicity was also prevented by the transgenic expression of spermine synthase in the Gy background.     

Sequential protein kinase C (PKC)-dependent and PKC-independent protein kinase D catalytic activation via Gq-coupled receptors: differential regulation of activation loop Ser(744) and Ser(748) phosphorylation

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸ revealed striking PKC-independent phosphorylation of Ser⁷⁴⁸ as well as Ser⁷⁴⁴ phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors (1). Many of these agonists bind to G protein-coupled receptors (GPCRs),⁴ activate heterotrimeric G proteins and stimulate isoforms of the phospholipase C family, including β, γ, δ, and ε (reviewed in Refs. 1 and 2). Activated phospholipase Cs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate mobilizes Ca²⁺ from intracellular stores (3, 4) whereas DAG directly activates the classic (α, β, and γ) and novel (δ, ε, η, and θ) isoforms of PKC (5–7). Although it is increasingly recognized that each PKC isozyme has specific functions in vivo (5–8), the mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely defined.PKD, also known initially as PKCμ (9, 10), and two recently identified serine protein kinases termed PKD2 (11) and PKCν/PKD3 (12, 13), which are similar in overall structure and primary amino acid sequence to PKD (14), constitute a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase group (15) and separate from the previously identified PKCs (14). Salient features of PKD structure include an N-terminal regulatory region containing a tandem repeat of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain) that confers high affinity binding to phorbol esters and DAG (9, 16, 17), followed by a pleckstrin homology (PH) domain that negatively regulates catalytic activity (18, 19). The C-terminal region of the PKDs contains its catalytic domain, which is distantly related to Ca²⁺-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity maintained by the N-terminal domain, which represses the catalytic activity of the enzyme by autoinhibition. Consistent with this model, deletions or single amino acid substitutions in the PH domain result in constitutive kinase activity (18–20). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (21). In response to cellular stimuli, PKD is converted from a low activity form into a persistently active form that is retained during isolation from cells, as shown by in vitro kinase assays performed in the absence of lipid co-activators (21, 22). PKD activation has been demonstrated in response to engagement of specific GPCRs either by regulatory peptides (23–30) or lysophosphatidic acid (27, 31, 32); signaling through G_q, G₁₂, G_i, and Rho (27, 31–34); activation of receptor tyrosine kinases, such as the platelet-derived growth factor receptor (23, 35, 36); cross-linking of B-cell receptor and T-cell receptor in B and T lymphocytes, respectively (37–40); and oxidative stress (41–44).Throughout these studies, multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF 109203X or Gö 6983) that do not directly inhibit PKD catalytic activity (21, 22), implying that PKD activation in intact cells is mediated, directly or indirectly, through PKCs. In line with this conclusion, cotransfection of PKD with active mutant forms of “novel” PKCs (PKCs δ, ε, η, and θ) resulted in robust PKD activation in the absence of cell stimulation (21, 44–46). Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade in response to multiple GPCR agonists in a broad range of cell types, including normal and cancer cells (reviewed in Ref. 14). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as the activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (reviewed in Ref. 14). Collectively, these findings demonstrated the existence of rapidly activated PKC-PKD protein kinase cascade(s) and raised the possibility that some PKC-dependent biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular activities and processes, including signal transduction (30, 47–49), chromatin modification (50), Golgi organization and function (51, 52), c-Jun function (47, 53, 54), NFκB-mediated gene expression (43, 55, 56), and cell survival, migration, and differentiation and DNA synthesis and proliferation (reviewed in Ref. 14). Thus, mounting evidence indicates that PKD has a remarkable diversity of both its signal generation and distribution and its potential for complex regulatory interactions with multiple downstream pathways, leading to multiple responses, including long term cellular events. Despite increasing recognition of its importance, very little is known about the mechanism(s) of sustained PKD activation as opposed to the well documented rapid, PKC-dependent PKD activation.The results presented here demonstrate that prolonged GPCR-induced PKD activation is mediated by sequential PKC-dependent and PKC-independent phases of regulation. We report here, for the first time, that PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies expand previous models of PKD regulation by identifying a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.     

Structure and Orientation of Troponin in the Thin Filament

The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-Å axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process with implications ranging from cell migration to muscle contraction. Skeletal and cardiac muscle thin filaments consist of actin and the regulatory proteins troponin and tropomyosin. Contraction is initiated by release of Ca²⁺ into the sarcomere and the consequent binding of Ca²⁺ to regulatory sites on troponin. Troponin is believed to undergo a conformational change leading to an azimuthal movement of tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP, and generate force. The molecular basis by which troponin acts to regulate muscle contraction is only partly understood. It is essential that the structure of troponin in the thin filament at high and low Ca²⁺ is determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972 (1). In this structure each tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å stagger between troponin molecules. The 7-Å tropomyosin structure (2), the atomic model of F-actin (3), and the troponin “core domain” (4) have recently been used to generate atomic models of the thin filament in low and high Ca²⁺ states (5). While the position of troponin in these models was constrained by known distance measurements between filament components, the exact arrangement of the complex on the filament has not been determined a priori. Although recently published crystal structures of partial troponin complexes (4, 6) have provided valuable insights into the arrangement of the globular head or core domain, the complex in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended tail (7). The globular core contains the Ca²⁺-binding subunit (TnC),² the inhibitory subunit (TnI), and the C-terminal part (residues 156–262) of the tropomyosin-binding subunit (TnT). The extended tail consists of the N-terminal part of TnT (residues 1–155). A structural rearrangement associated with Ca²⁺ dissociation from the troponin core has been observed (4) such that the helix connecting the two domains of TnC collapses, releasing the TnI inhibitory segment. It is postulated that the TnI inhibitory segment then becomes able to bind actin, in so doing biasing tropomyosin (8). To understand properly how Ca²⁺ binding to TnC leads to movement of tropomyosin, it is necessary to determine a high resolution structure of troponin attached to the thin filament, allowing unambiguous docking of the available crystal structures and direct observation of any changes at a molecular level caused by Ca²⁺ binding.Direct visualization of the thin filament is possible using electron microscopy. Tropomyosin strands have been resolved in the low and high Ca²⁺ states confirming the movement of tropomyosin and the steric blocking model (9, 10). Until recently the actin helical repeat has been imposed in the majority of reconstructions of the thin filament causing artifacts. Helical averaging using the actin repeat spreads troponin density over every actin monomer, which prevents the detailed position and shape of the troponin complex from being found (11). It is possible to avoid this effect by applying a single particle approach. Individual filament images are divided into segments and each segment treated as a particle. Three-dimensional reconstruction may then be carried out by single particle techniques of alignment, classification (12, 13), Euler angle assignment (14–16) and exact filter back-projection (17, 18).Two forms of single particle analysis have emerged: helical single particle analysis (19), where the determined helical symmetry is applied to the final reconstruction, and non-helical single particle analysis, which treats the complex as a truly asymmetric particle. Helical single particle analysis has been used to successfully reconstruct a myosin containing invertebrate thick filament to a resolution of 25 Å (20), and non-helical single particle analysis has been applied to the vertebrate skeletal muscle thick filament allowing azimuthal perturbations of the myosin heads to be observed (21).Model-based single particle image processing methods have recently been applied to the structural analysis of the vertebrate (5, 22, 23) and the insect thin filament (24). We have deliberately avoided starting with a model and any potential model bias by using a reference-free alignment procedure. The adaptation of conventional procedures and their application to the structural study of the muscle thin filament has been documented (25).     

Oligomerization of the Macrophage Mannose Receptor Enhances gp120-mediated Binding of HIV-1

C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.The mannose receptor (MR)² is a C-type lectin receptor that is expressed on the surface of a variety of cells, including immature monocyte-derived dendritic cells (MDDC), dermal dendritic cells, macrophages, and hepatic endothelial cells. It is a multifunctional protein, involved in antigen recognition and internalization during the early stages of the innate immune response (1) as well as physiological clearance of the endogenous pituitary hormones lutropin and thyrotropin (2, 3). Recognition of foreign antigens occurs via mannose, fucose, and GlcNAc residues (4, 5), which are generally not found as terminal residues on mammalian glycoproteins but are highly abundant on surface proteins of pathogens such as the HIV-1 envelope gp120 (6, 7). Once bound, pathogens can be internalized by endocytosis or phagocytosis, where they are targeted to lysosomes for proteolytic degradation and presentation on major histocompatibility complex class II (8). In immature DCs, soluble recombinant HIV envelope proteins are processed by this pathway, initially binding to both dendritic cell-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR but not DC-SIGN in lysosomes (9). Furthermore, in immature DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a unique vesicular compartment that co-localizes with tetraspanin proteins such as CD81 (10, 11). Recently, this compartment has been shown to be continuous with the plasma membrane (11) and does not represent a continuation of the endolysosomal network. Interestingly, this compartment can translocate virus from DCs to CD4 T cells, upon the formation of a virological synapse (10–12). Although viral uptake can occur in DCs independent of HIV env (2), the efficiency of HIV binding and uptake is greatly enhanced by the presence of C-type lectin-env interactions. At least initial binding to DC-SIGN (and most likely also MR) is required for T cell trans-infection (13).Structurally, the extracellular domain of MR consists of an N-terminal cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and eight carbohydrate recognition domains (CRD) on a single polypeptide backbone (1). Of the eight CRDs, CRD 4–8 have been shown to be required for high affinity binding of ligands containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having demonstrable monosaccharide binding in isolation (14). Binding and release of ligand within the low pH environment of the endolysosomal compartment are also Ca²⁺-dependent. Acid-induced removal of Ca²⁺ binding in CRD 4 and 5 was shown to cause a conformational rearrangement of the domain, resulting in a loss of carbohydrate binding activity (15). In contrast, binding of sulfated carbohydrates to the Cys-RD appears to be Ca²⁺-independent as no Ca²⁺-binding sites were observed in its crystal structure (2, 16).Oligomerization of CLRs such as DC-SIGN (17), Langerin (18), and mannose-binding protein (19) has been reported to be essential for binding of oligosaccharide-bearing ligands. Early studies on MR suggested that it exists solely as a monomeric molecule and that clustering of multiple CRDs within the single polypeptide backbone was necessary for high affinity binding of oligosaccharide moieties (20). However, more recent studies have shown that dimerization is possible in the presence of Ca²⁺ (21) and that an equilibrium may exist between monomeric and dimeric forms on the cell surface (22). It is currently unclear what effect dimerization has on ligand binding to the CRDs; however, there is evidence that dimerization of MR is required for high affinity binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate (GalNAc-4-SO₄) such as lutropin and thyrotropin (22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR have used solubilized protein from cell lysates (20) or purified recombinant fragments (21). Because the membrane microenvironment can influence protein associations, soluble forms of MR may not necessarily be a true model of the quaternary structure and function of the native protein. Here, we used a well established method of cross-linking (23) on MDDCs, monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to preserve lateral protein-protein interactions between MR on the cell surface prior to solubilization. Mass spectrometry analysis of affinity-purified complexes showed they were homo-oligomers, and further resolution of the complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates that they are dimers. Dimerization of MR was also found to be essential for binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral particles. Persistence of monomeric gp120 and trimeric gp140 binding to dimeric MR in the presence of EGTA and various CRD and other inhibitors, however, suggested that gp120-mediated HIV-1 binding is not Ca²⁺-dependent and that at least binding probably occurs to both Ca²⁺-dependent and -independent CRDs and also the Cys-RD.     

A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca²⁺ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [¹²⁵I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca²⁺. This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca²⁺ and calmodulin decreases by approximately the same amount. However, incubation with Ca²⁺ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [¹²⁵I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E₁ conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca²⁺ + C28 for SERCA and Ca²⁺ alone for PMCA), and one in which the enzyme is fully active (Ca²⁺ for SERCA and Ca²⁺-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.Although membrane proteins constitute more than 20% of the total proteins, the structure of only few of them is known in detail. An important group of integral membrane proteins are ion-motive ATPases. These proteins belong to the family of P-type ATPases, which share in common the formation of an acid-stable phosphorylated intermediate as part of its reaction cycle. Crystallographic information is available for a few members of this family. There are several crystal structures of the Ca²⁺ pump of sarcoplasmic reticulum (SERCA)² revealing different conformations (1–5), and recently, crystal structures of the H⁺-ATPase (6) and of the Na,K-ATPase were reported as well (7).We are interested in obtaining structural information about the plasma membrane calcium pump (PMCA). This pump is an integral part of the Ca²⁺ signaling mechanism (8). It is highly regulated by calmodulin, which activates this protein by binding to an auto-inhibitory region and changing the conformation of the pump from an inhibited state to an activated one (8, 9). Crystallization of PMCA is particularly challenging because there is no natural source from which this protein can be obtained in large quantities. Moreover, the presence of several isoforms in the same tissue further complicates efforts to obtain a homogeneous sample suitable for crystallization.Information about the structure and assembly of the transmembrane domain of an integral membrane protein can also be obtained from the analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between PMCA and the surrounding phospholipids under different experimental conditions that lead to known conformations. We employed the photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) that has been previously used to analyze lipid-protein interfaces (10–12). This reagent is located in the phospholipidic milieu, and upon photolysis it reacts indiscriminately with its molecular neighbors. It is thus possible to directly analyze the interaction between a membrane protein and lipids belonging to its immediate environment (13–15). By measuring the amount of labeling of SERCA in conditions that promote conformations for which there are well resolved crystal structures, we were able to validate this photolabeling approach as a convenient tool for analyzing conformational changes within transmembrane regions. Furthermore, using this technique on PMCA and comparing the results obtained for SERCA, we were able to draw structural conclusions about these proteins under activated and inhibited states.