Post-Translational Modifications of β Subunits of Voltage-Dependent Calcium Channels

Different post-translational modifications of Ca channel subunits have been identified. Recent studies have characterized the palmitoylation of the Ca channel _2a subunit, as well as one effect of this modification on channel function. The potential importance of palmitoylation on other channel properties is discussed. Other studies have addressed the role of phosphorylation of subunits in the regulation of voltage-dependent Ca channels. Phosphorylation of subunits by second messenger-activated protein kinases, as well as by unidentified protein kinases, may affect interactions between channel subunits and other aspects of channel function. The differential modification of Ca channel subunit isoforms by post-translational events likely results in diversely regulated channels with unique properties.     

The Voltage-Gated Calcium Channel γ Subunits: A Review of the Literature

Members of the voltage-gated calcium channel y subunit gene family (Cacng), have been rapidly discovered since the discovery of the identification of the mouse gamma2 gene (Cacng2) and its association with the stargazer mutant mouse line. The fact that this mutant mouse line exists has allowed researchers to gain insights into the function of the gamma2 subunit. For example, stargazer mice have elevated levels of neuropeptide Y production, very low cerebellar brain derived neurotrophic factor production, and diminished cerebellar GABAA alpha6 and beta3 production. Study of this mutant mouse line has also revealed that the gamma2 subunit is involved in AMPA receptor trafficking and targeting to the synaptic membrane. For the most part, the effect of gamma2 subunits on the electrophysiology of voltage-gated calcium channels is to downregulate calcium channel activity by causing a hyperpolarizing shift in the inactivation curve. This finding and the association of these subunits with AMPA receptor trafficking has led some researchers to question the actual role of the gamma subunits. This article reviews the discovery, cellular localization, tissue distribution, and function of the eight members of the Cacng family.     

β Subunits of Voltage-Gated Calcium Channels

Calcium channel beta subunits have marked effects on the trafficking and on several of the biophysical properties of all high voltage activated calcium channels. In this article I shall review information on the different genes, on the structure of the beta subunits, and on their differential expression and post-translational modification. Their role in trafficking and assembly of the calcium channel heteromultimer will be described, and I will then review their effects on voltage-dependent and kinetic properties, stressing the differences between palmitoylated beta2a and the other beta subunits. Evidence for effects on calcium channel pharmacology will also be examined. I shall discuss the hypothesis that beta subunits can bind reversibly to calcium channels, and examine their role in the G protein modulation of calcium channels. Finally, I shall describe the consequences of knock-out of different beta subunit genes, and describe evidence for the involvement of beta subunits in disease.     

Structural Basis for Calcium and Magnesium Regulation of a Large Conductance Calcium-activated Potassium Channel with β1 Subunits

Large conductance Ca²⁺- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary β subunits, play important roles in diverse physiological activities. The β1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca²⁺ sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and β1 remains elusive. Using macroscopic ionic current recordings in various Ca²⁺ and Mg²⁺ concentrations, we identified two binding sites on the cytosolic N terminus of β1, namely the electrostatic enhancing site (mSlo1(K392,R393)-β1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-β1(L5,V6,M7)), passing the physical force from the Ca²⁺ bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg²⁺ sensitivity. A comprehensive structural model of the BK(mSlo1 α-β1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.     

Calcium Channel α2δ Subunits: Differential Expression, Function, and Drug Binding

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.     

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca²⁺ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α_1s or Ca_V1.1, α₂, β_1a, δ1, and γ), we created transgenic mice expressing a recombinant β_1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α₂-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca_V1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α_1s subunit to the membrane and its suggested role in excitation-contraction coupling.     

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

Background

The Ca_vβ subunits of high voltage-activated Ca²⁺ channels control the trafficking and biophysical properties of the α₁ subunit. The Ca_vβ-α₁ interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that βs regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the α-interaction domain (AID) be a rigid structure.

Methodology/Principal Findings

The present study tests this hypothesis by altering the flexibility and orientation of this region in α₁2.2, then testing for Ca_vβ regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished β2a and β3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of β2a to produce non-inactivating currents. Orientation of Ca_vβ with respect to α₁2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca_vβ subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca_vβ subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms β-sheet. The orientation of β with respect to α was confirmed by the bimolecular fluorescence complementation assay.

Conclusions/Significance

These results show that the orientation of the Ca_vβ subunit relative to the α₁2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca_vβ to regulate channel activity.     

Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.     

Voltage-Operated Calcium Channel Heterogeneity in Pancreatic β Cells: Physiopathological Implications

Voltage-operated calcium channels play crucial roles in stimulus-secretion coupling in pancreatic beta cells. A growing body of evidence indicates that these channels in beta cells are heterogeneous. In particular, not all the high-threshold calcium channels expressed belong to the best known L-type. In rat insulinoma cells, for example, L, N, and P/Q-type channels are present, while in human beta cells L-type and P/Q-type dominate. Where present, N-type and P/Q-type channels participate, alongside with the dominant L-type, in the control of sugar- or depolarization-induced hormone release. Distinct biophysical properties and selective modulation of the channel subtypes are likely to play important physiological roles. T-type channels are involved in beta cell apoptosis, while calcium channel autoantibodies recognizing high-threshold channels in beta cells, have been described both in neurological and diabetic patients. Subtype-selective calcium channel drugs have the potential for being beneficial in beta cell pathological states.     

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.     

The HOOK-Domain Between the SH3- and the GK-Domains of CaVβ Subunits Contains Key Determinants Controlling Calcium Channel Inactivation

Ca_Vβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (5th) β-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Ca_Vβ subunits. Here we have been guided by the recent structural studies of Ca_Vβ subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Ca_Vβ subunits in the modulation of inactivation of N-type calcium channels. We have co-expressed the β subunit constructs with Ca_V2.2 and α2δ-2, using the N-terminally palmitoylated β2a subunit, because it supports very little voltage-dependent inactivation, and making comparisons with β1b domains. Deletion of the variable region of the β2a HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the β2a palmitoylation motif further enhanced inactivation. The isolated GK-domain of β2a alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the β2a-GK-domain construct was extended proximally, by including the HOOK-domain and the ε-strand of the SH3-domain, inactivation was about 4 fold slower than in the absence of the HOOK domain. When the SH3-domain of β2a truncated prior to the HOOK-domain was co-expressed with the (HOOK+εSH3+GK)-domain of β2a, all the properties of β2a were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+εSH3+GK)-β2a construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.     

Nonchannel Functions of the Calcium Channel γ Subunit: Insight from Research on the Stargazer Mutant

Voltage-dependent calcium channels (VDCC) are essential regulators of intracellular calcium concentration, which in turn influences a broad spectrum of cellular functions especially in neurons. Identification of several calcium channel mutations as the cause of neurological disorders in human and mouse indicates the importance of the integrity of these channels to neuronal function. Studies of mutant mice, each carrying a disrupted gene of a different VDCC subunit, have revealed many unexpected roles of these molecules and have significantly advanced our knowledge of subunit function in the last few years. This review addresses recent discoveries of the function of the gamma2 subunit, also named stargazin, with special emphasis on roles other than calcium conductance.     

Isolation of α and β Brain Tubulin Subunits after Alkaline Treatment of the Protein

We demonstrate here that brain purified tubulin can be dissociated into and subunits at pH > 10 and that the subunits can be separated by using the Triton X-114 phase separation system. After phase partition at pH > 10, tubulin but not tubulin behaves as a hydrophobic compound appearing in the detergent rich phase. After three extractions of the alkaline aqueous phase with Triton X-114, about 90% of the tubulin was recovered in the detergent rich phase. The hydrophobic behavior observed for tubulin after its dissociation at pH 11.5 was not due to an irreversible change of the protein, because when the detergent rich phase containing tubulin was diluted with a buffer solution at pH 7.3 and the solution allowed to partition again, -tubulin is recovered in the aqueous phase. The detergent in the aqueous phase of the and tubulin preparations can be removed up to 90% by 12 h dialysis. The and subunits of tubulin from kidney and liver behave, in this phase separation system, like those of brain tubulin.     

A Novel Biological Activity of Praziquantel Requiring Voltage-Operated Ca2+ Channel β Subunits: Subversion of Flatworm Regenerative Polarity

Background

Approximately 200 million people worldwide harbour parasitic flatworm infections that cause schistosomiasis. A single drug—praziquantel (PZQ)—has served as the mainstay pharmacotherapy for schistosome infections since the 1980s. However, the relevant in vivo target(s) of praziquantel remain undefined.

Methods and Findings

Here, we provide fresh perspective on the molecular basis of praziquantel efficacy in vivo consequent to the discovery of a remarkable action of PZQ on regeneration in a species of free-living flatworm (Dugesia japonica). Specifically, PZQ caused a robust (100% penetrance) and complete duplication of the entire anterior-posterior axis during flatworm regeneration to yield two-headed organisms with duplicated, integrated central nervous and organ systems. Exploiting this phenotype as a readout for proteins impacting praziquantel efficacy, we demonstrate that PZQ-evoked bipolarity was selectively ablated by in vivo RNAi of voltage-operated calcium channel (VOCC) β subunits, but not by knockdown of a VOCC α subunit. At higher doses of PZQ, knockdown of VOCC β subunits also conferred resistance to PZQ in lethality assays.

Conclusions

This study identifies a new biological activity of the antischistosomal drug praziquantel on regenerative polarity in a species of free-living flatworm. Ablation of the bipolar regenerative phenotype evoked by PZQ via in vivo RNAi of VOCC β subunits provides the first genetic evidence implicating a molecular target crucial for in vivo PZQ activity and supports the ‘VOCC hypothesis’ of PZQ efficacy. Further, in terms of regenerative biology and Ca²⁺ signaling, these data highlight a novel role for voltage-operated Ca²⁺ entry in regulating in vivo stem cell differentiation and regenerative patterning.     

Diversity and Developmental Expression of L-type Calcium Channel ��2 Proteins and Their Influence on Calcium Current in Murine Heart

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVβ2 proteins are the major CaVβ components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVβ3 proteins are also detectable. At least two CaVβ2 variants of ∼68 and ∼72 kDa are expressed. To identify the underlying CaVβ2 variants, cDNA libraries were constructed from poly(A)⁺ RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVβ2 cDNA clones coding for four types of CaVβ2 proteins only differing in their 5′ sequences. CaVβ2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVβ2 protein pattern in vitro. Significant L-type Ca²⁺ currents (I_Ca) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVβ2. Current kinetics were determined by the type of CaVβ2 protein, with the ∼72-kDa CaVβ2a-N1 shifting the activation of I_Ca significantly to depolarizing potentials compared with the other CaVβ2 variants. Inactivation of I_Ca was accelerated by CaVβ2a-N1 and -N4, which also lead to slower activation compared with CaVβ2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVβ2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVβ2a-N1-induced changes of I_Ca kinetics might be essential in embryonic heart.Cardiac contractions require Ca²⁺ influx in cardiomyocytes from the extracellular fluid, which leads to Ca²⁺ release from the sarcoplasmic reticulum via ryanodine receptors (1).This Ca²⁺-induced Ca²⁺ release (CICR)⁴ causes a marked increase in intracellular Ca²⁺ concentration for short periods of time and underlies cardiac contraction (2, 3). The Ca²⁺ influx into cardiac myocytes is mediated by high voltage-activated L-type Ca²⁺ channels (LTCCs), which are heteromultimeric complexes comprised predominantly of the pore-forming CaVα₁ subunit and the auxiliary CaVβ subunit (4). In heart, the principal CaVα₁ subunit, CaVα₁c (CaV1.2), is encoded by the Cacna1C gene (5). Four genes (Cacnb1-4) encoding CaVβ subunits have been identified that are expressed in the heart of different species including human, rabbit, and rat (6, 7, 8).CaVβ proteins are ∼500 amino acid cytoplasmic proteins that bind to the CaVα₁ I-II intracellular loop (9) and affect channel gating properties (4), trafficking (10, 11), regulation by neurotransmitter receptors through G-protein βγ subunit activation (12), and sensitivity to drugs (13). The CaVβ primary sequence encodes five domains, arranged V1-C1-V2-C2-V3. V1, V2, and V3 are variable domains, whereas C1 and C2 are conserved (14). Structural studies reveal that C1 and C2 form a SH3 domain (Src homology 3 domain) and a NK domain (nucleotide kinase domain), respectively (15). Although C1-V2-C2 makes the CaVβ core, in heart the V1 region appears critical for the kinetics of I_Ca and heart function. Accordingly a mutation in the V1 region of the Cacnb2 gene was recently identified as an underlying cause of Brugada syndrome (16).In mice-targeted deletion of the Cacnb2 gene (17) but not of Cacnb1 (18), Cacnb3 (19, 20), or Cacnb4 (21) leads to a morphologically and functionally compromised heart, which causes severe defective remodeling of intra- and extra-embryonic blood vessels and death at early embryonic stages both when the Cacnb2 gene was targeted globally or in a cardiac myocyte-specific way (17). Although these results point to an essential role of CaVβ2 for I_Ca and cardiac function, the existence of various CaVβ2 splice variants and heterogeneity of the expressed CaVβ2 proteins require further studies on the subunit composition of LTCCs in the mouse heart. In addition and in view of the growing number of preclinical studies using mouse models carrying definite Ca²⁺ channel subunits as transgenes in heart tissue, the identification of the relevant gene products underlying the endogenous mouse cardiac L-type channel is essential. Recent mouse models (e.g. 22, 23, 24) carrying a rat CaVβ2 splice variant (“rat CaVβ2a”) (25) expressed in rat and rabbit brain (26), but not in rabbit heart (26), have only escalated this requirement, because it has never been shown that the mouse orthologue of this variant is endogenously expressed in the mouse heart.So far, five CaVβ2 variants varying only in the V1 domain have been identified from different species (25, 27, 28) and in human heart these variants have been obtained mainly by RT-PCR approaches (29, 30). In contrast, there is little information on the CaVβ proteins present in mouse heart, their respective splice variants, and expression ratios. We therefore started to study CaVβ expression in the murine heart using Western blots and cDNA cloning and to reveal their functional impact on LTCCs formed by the murine CaV1.2 protein.     

Functions of the ��, ��, and �� Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α₂β₂γ₂ hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α₂β₂γ₂ and α₂β₂ forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the acid hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner.     

The Functional Role of β Subunits in Oligomeric P-Type ATPases

Na,K-ATPase and gastric and nongastric H,K-ATPases are the only P-type ATPases of higher organisms that are oligomeric and are associated with a subunit, which is obligatory for expression and function of enzymes. Topogenesis studies suggest that subunits have a fundamental and unique role in K⁺-transporting P-type ATPases in that they facilitate the correct membrane integration and packing of the catalytic subunit of these P-type ATPases, which is necessary for their resistance to cellular degradation, their acquisition of functional properties, and their routing to the cell surface. In addition to this chaperone function, subunits also participate in the determination of intrinsic transport properties of Na,K- and H,K-ATPases. Increasing experimental evidence suggests that assembly is a highly ordered, isoform-specific process, which is mediated by multiple interaction sites that contribute in a coordinate, multistep process to the structural and functional maturation of Na,K- and H,K-ATPases.