Lipids Modulate the Increase of BK Channel Calcium Sensitivity by the β1 Subunit

Co-expression of the auxiliary β1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of β1′s interaction with α. We take a novel approach, exploring whether β1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+β1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14∶1), C (18∶1), C (22∶1) and C (24∶1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the β1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca²⁺ sensitivity by β1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of β1 reduces BK activity in thin bilayers of PC 14∶1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18∶1, PC 22∶1 and PC 24∶1 bilayers. These data suggest that auxiliary β1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions.     

In Vivo and Ex Vivo Evaluation of L-Type Calcium Channel Blockers on Acid β-Glucosidase in Gaucher Disease Mouse Models

Gaucher disease is a lysosomal storage disease caused by mutations in acid β-glucosidase (GCase) leading to defective hydrolysis and accumulation of its substrates. Two L-type calcium channel (LTCC) blockers—verapamil and diltiazem—have been reported to modulate endoplasmic reticulum (ER) folding, trafficking, and activity of GCase in human Gaucher disease fibroblasts. Similarly, these LTCC blockers were tested with cultured skin fibroblasts from homozygous point-mutated GCase mice (V394L, D409H, D409V, and N370S) with the effect of enhancing of GCase activity. Correspondingly, diltiazem increased GCase protein and facilitated GCase trafficking to the lysosomes of these cells. The in vivo effects of diltiazem on GCase were evaluated in mice homozygous wild-type (WT), V394L and D409H. In D409H homozygotes diltiazem (10 mg/kg/d via drinking water or 50–200 mg/kg/d intraperitoneally) had minor effects on increasing GCase activity in brain and liver (1.2-fold). Diltiazem treatment (10 mg/kg/d) had essentially no effect on WT and V394L GCase protein or activity levels (<1.2-fold) in liver. These results show that LTCC blockers had the ex vivo effects of increasing GCase activity and protein in the mouse fibroblasts, but these effects did not translate into similar changes in vivo even at very high drug doses.     

A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.     

Aminopyridines Potentiate Synaptic and Neuromuscular Transmission by Targeting the Voltage-activated Calcium Channel �� Subunit

Aminopyridines such as 4-aminopyridine (4-AP) are widely used as voltage-activated K⁺ (Kv) channel blockers and can improve neuromuscular function in patients with spinal cord injury, myasthenia gravis, or multiple sclerosis. Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca²⁺ channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, α-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca²⁺ currents primarily through the intracellular β₃ subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels.     

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

γ-Aminobutyric acid type A (GABA_A) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA_A receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABA_A receptors by glycosylation.     

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.     

Phosphoinositide 3-Kinase C2�� Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2β. Dbl was co-immunoprecipitated with PI3KC2β in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2β in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2β strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2β over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2β expression. Furthermore, increased expression of PI3KC2β protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2β modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.     

Allelic Variants Interaction of CLOCK Gene and G‐Protein β3 Subunit Gene with Diurnal Preference

The 3111 C/T single nucleotide polymorphism (SNP) in the CLOCK gene and the 825C/T SNP in the G‐protein β3 subunit gene (GNB3) have been reported to influence diurnal preference. This study has attempted to characterize the association between the CLOCK gene and GNB3 polymorphisms and diurnal preference in healthy Korean college students. All subjects completed the 13‐item Composite Scale for Morningness (CSM). The interaction between the 3111 C/T SNP in the CLOCK gene and the 825 C/T SNP in the GNB3 gene significantly influenced diurnal preference, according to the CSM Performance subscore (F=10.94, p=0.001). However, when the different polymorphisms of the two genes were analyzed independently, no direct correlations with diurnal preference were detected. The CLOCK gene 3111 C/T SNP and GNB3 gene 825 C/T SNP were found to manifest a gene‐gene interaction that affects diurnal preference.     

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.     

Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.     

TGF-? Regulates Enamel Mineralization and Maturation through KLK4 Expression

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.     

Single-Channel Monitoring of Reversible L-Type Ca Channel CaVα1-CaVβ Subunit Interaction

Voltage-dependent Ca²⁺ channels are heteromultimers of Ca_Vα₁ (pore), Ca_Vβ- and Ca_Vα₂δ-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, Ca_Vβ-isoforms affect gating differentially, and we chose two extremes (Ca_Vβ_1a and Ca_Vβ_2b) regarding single-channel open probability to address this question. HEK293α_1C cells expressing the Ca_V1.2 subunit were transiently transfected with Ca_Vα₂δ1 alone or with Ca_Vβ_1a, Ca_Vβ_2b, or (2:1 or 1:1 plasmid ratio) combinations. Both Ca_Vβ-subunits increased whole-cell current and shifted the voltage dependence of activation and inactivation to hyperpolarization. Time-dependent inactivation was accelerated by Ca_Vβ_1a-subunits but not by Ca_Vβ_2b-subunits. Mixtures induced intermediate phenotypes. Single channels sometimes switched between periods of low and high open probability. To validate such slow gating behavior, data were segmented in clusters of statistically similar open probability. With Ca_Vβ_1a-subunits alone, channels mostly stayed in clusters (or regimes of alike clusters) of low open probability. Increasing Ca_Vβ_2b-subunits (co-)expressed (1:2, 1:1 ratio or alone) progressively enhanced the frequency and total duration of high open probability clusters and regimes. Our analysis was validated by the inactivation behavior of segmented ensemble averages. Hence, a phenotype consistent with mutually exclusive and dynamically competing binding of different Ca_Vβ-subunits is demonstrated in intact cells.     

Calretinin Regulates Ca2+-dependent Inactivation and Facilitation of Cav2.1 Ca2+ Channels through a Direct Interaction with the α12.1 Subunit

Voltage-gated Ca_v2.1 Ca²⁺ channels undergo dual modulation by Ca²⁺, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca²⁺-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α₁2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α₁2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca²⁺-dependent. α₁2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca_v2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca_v2.1 CDI and increased CDF. CR binding to α₁2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α₁2.1 with that from the Ca_v1.2 α₁ subunit (α₁1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α₁2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca_v2.1, which may have major consequences for Ca²⁺ signaling and neuronal excitability.