Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits

Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the selectivity of the actions of CaMKII among Ca2+ channel beta subunit isoforms. CaMKII phosphorylates the beta1b, beta2a, beta3, and beta4 isoforms with similar initial rates and final stoichiometries of 6-12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to beta1b and beta2a with a similar apparent affinity but does not bind to beta3 or beta4. Prephosphorylation of beta1b and beta2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in beta2a are highly conserved in beta1b but are different in beta3 and beta4. Site-directed mutagenesis of this domain in beta2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-beta2a complexes in vitro and reduces interactions of CaMKII with beta2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with beta2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca2+ channel beta subunits with CaMKII.     

Inhibition of voltage-gated calcium channels by sequestration of beta subunits

The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.     

A family of gamma-like calcium channel subunits

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.     

Mutations in voltage-gated L-type calcium channel: implications in cardiac arrhythmia

The voltage-gated L-type calcium channel (LTCC) is essential for multiple cellular processes. In the heart, calcium influx through LTCC plays an important role in cardiac electrical excitation. Mutations in LTCC genes, including CACNA1C, CACNA1D, CACNB2 and CACNA2D, will induce the dysfunctions of calcium channels, which result in the abnormal excitations of cardiomyocytes, and finally lead to cardiac arrhythmias. Nevertheless, the newly found mutations in LTCC and their functions are continuously being elucidated. This review summarizes recent findings on the mutations of LTCC, which are associated with long QT syndromes, Timothy syndromes, Brugada syndromes, short QT syndromes, and some other cardiac arrhythmias. Indeed, we describe the gain/loss-of-functions of these mutations in LTCC, which can give an explanation for the phenotypes of cardiac arrhythmias. Moreover, we present several challenges in the field at present, and propose some diagnostic or therapeutic approaches to these mutation-associated cardiac diseases in the future.     

Interferon-gamma activates the voltage-gated calcium channel in RPMI 4788 cells

Interferon (IFN) increases cytoplasmic free calcium ([Ca2+]i) in RPMI 4788 cells, a human colon cancer cell line. Addition of IFN to the cells loaded with Fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after IFN stimulation. A cyanine dye, dis-C3- (5) was used to determine the effects of IFN on the membrane potential in cancer cells. The depolarization was seen with IFN-gamma, but not with IFN-beta. These results taken together, suggest that the IFN-gamma and -beta induced [Ca2+]i mobilization are clearly different in their dependence on Ca2+ entry through voltage-gated Ca2+ channels.     

Towards a unified nomenclature describing voltage-gated calcium channel genes

A unified nomenclature for describing voltage-gated calcium channel genes is proposed. The terminology has been approved by the HUGO/GDB nomenclature committee. Received: 5 February 1997 / Accepted: 4 April 1997     

Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca(V)s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca(2+)/calmodulin (Ca(2+)/CaM) and a Ca(V) channel consensus isoleucine-glutamine (IQ) motif: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the high-resolution structure of the Ca(2+)/CaM-Ca(V)1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca(2+)/CaM lobes through sets of conserved 'aromatic anchors.' Ca(2+)/N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca(2+)/N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca(2+)/N lobe aromatic anchors uncovers their role in Ca(V)1.2 CDF. Together, our data suggest that Ca(V) subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca(V)s.     

Meiosis reinitiation of mussel oocytes involves L-type voltage-gated calcium channel

In the present work we assessed the involvement of L-type voltage opening Ca²⁺ channels in KCl-induced meiosis reinitiation of metaphase-arrested blue mussel (Mytilus galloprovincialis) oocytes by performing binding assays with a tritiated dihydropyridine analog (+)PN 200110. Our data reveal the existence of a single class of dihydropyridine receptors in plasma membrane-enriched rough microsome preparations of mussel oocytes. The apparent affinity (K_d) of characterized receptors equals 1.32 ± 0.21 μM while their maximal binding capacity (B_max) is 620 ± 150 pmol/mg protein. The comparison of the rank order of potency of analogs tested to: 1) inhibit [(+)-l³H]PN 200110 specific binding and 2) block KCl-induced meiosis reinitiation pointed to the pharmacological profile similar to but not identical with those previously described for mammalian dihydropyridine receptors. The efficiencies of all antagonists tested are linearly related (r = 0.995) in binding- (inhibition of [(+)-l³H]PN 200110 specific binding) and biological (inhibition of meiosis reinitiation) assays thus arguing for functional involvement of L-type Ca²⁺ channels in oocyte activation. Reversibility of antagonist actions on meiosis reinitiation and dependence of receptor binding characteristics on a membrane polarization state further suggested such a role. J. Cell. Biochem. 64:152–160. © 1997 Wiley-Liss, Inc.     

Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [⁴⁵Ca²⁺] influx, which was due to the enhanced [⁴⁵Ca²⁺] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [³H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [³H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of α2/δ1 subunit. Similar changes in [³H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also α2/δ1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.     

Multiplicity of protein interactions and functions of the voltage-gated calcium channel beta-subunit

For a long time the auxiliary beta-subunit of voltage-gated calcium channels was thought to be engaged exclusively in the regulation of calcium channel function, including gating, intracellular trafficking, assembly and membrane expression. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins (MAGUK) that comprises a series of protein interaction motifs. Two such domains, a Src homology 3 and a guanylate kinase domain are present in the beta-subunit. Recently, it was shown that this subunit interacts with a variety of proteins and regulates diverse cellular processes ranging from gene expression to hormone secretion and endocytosis. In light of these new findings, the beta-subunit deserves to be promoted to the category of multifunctional regulatory protein. Some of these new functions support a tighter regulation of calcium influx through voltage-gated calcium channels and others apparently serve channel unrelated processes. Here we discuss a variety of protein-protein interactions held by the beta-subunit of voltage-gated calcium channels and their functional consequences. Certainly the identification of additional binding partners and effector pathways will help to understand how the different beta-subunit-mediated processes are interwoven.     

Labelling of the 3D structure of the cardiac L-type voltage-gated calcium channel

Voltage-gated calcium channels (VGCCs) regulate calcium influx into all excitable cells. In the heart, the main calcium channels are the L-type VGCCs (LTCCs). These are localised to the sarcolemmal membrane, and are hetero-oligomeric complexes comprised of three non-covalently associated polypeptides; alpha1 (Ca_V1.2), alpha2delta and beta. We recently reported the 3D structure for a monomeric form of the cardiac LTCC1 using electron microscopy and single particle analysis. We also determined the first medium/low resolution structure of a T-type voltage gated calcium channel (Ca_V3.1) polypeptide. We identified the transmembrane and cytoplasmic domains of the T-type channel using labelling studies to determine the position of the C-terminus. By modelling of the Ca_V3.1 structure (comparable at these resolutions to Ca_V1.2) into the cardiac LTCC volume, we were able to delineate the subunit boundaries of the cardiac LTCC, leading to a proposal for a putative orientation of the LTCC with respect to the membrane bilayer. We have now extended these studies to include labelling of the extracellular alpha2 polypeptide using affinity purified antibodies raised against the Von Willebrand Factor A (VWA) domain and calmodulin-gold labelling of the C-terminus of Ca_V1.2. These data provide further support for the proposed orientation of the 3D structure of the cardiac LTCC.     

Differential modulation of L-type calcium channel subunits by oleate

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca(2+) and increase voltage-gated Ca(2+) current in mouse beta-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca(2+) channels (LTCC) and demonstrate that beta-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type alpha(1)-subunits (beta-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet beta-subunits (ibeta(2a) or ibeta(3)). In COS-7 cells, elevations in intracellular Ca(2+) mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca(2+) current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca(2+) current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary beta-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca(2+) channel modulation. These results show for the first time that two L-type Ca(2+) channels expressed in beta-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.     

Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, α_1B and β₃ subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional ω-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of α_1B subunits was diffuse. The distribution of the α_1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 198–208, 1998     

Voltage-gated calcium channel subunits from platyhelminths: potential role in praziquantel action

Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+ -dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming alpha(1) subunit and other modulatory subunits, including the beta subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel beta subunits (Ca(v)betavar) found in these parasites. In particular, we examine the role these beta subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Ca(v)betavar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant beta subunits may thus represent a platyhelminth-specific gene family.     

Apo calmodulin binding to the L-type voltage-gated calcium channel Cav1.2 IQ peptide

The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Cav1.2 subunit has been shown to bind both calcium-loaded (Ca2+CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca2+CaM can bind to the intact channel.     

Specific subunits of voltage-gated sodium channel underlying the subthreshold membrane potential oscillations: Potential therapeutic targets for neuropathic pain

The treatment of neuropathic pain remains a major challenge to pain clinicians. Certain nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons may develop abnormal spontaneous activities following peripheral nerve injury, which is believed to be a major contributor to chronic pain. Subthreshold membrane potential oscillation (SMPO) observed in injured DRG neurons was reported to be involved in the generation of abnormal spontaneous activity. Tetrodotoxin-sensitive sodium (Na⁺) channels were testified to be involved in the generation of SMPO, but their specific subunits have not been clarified. We hypothesize that the subunits of voltage-gated sodium channel, Na_v1.3 and Na_v1.6, are involved in the generation of SMPO. An attempt to test this hypothesis may lead to a new therapeutic strategy for neuropathic pain.     

Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel

Highly purified L-type Ca(2+) channel complexes containing all five subunits (alpha(1), alpha(2), beta, gamma, and delta) and complexes of alpha(1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca(2+) channels and alpha(1)-beta complexes were aligned, classified and averaged. The alpha(1)-beta complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca(2+) channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca(2+) channel using a monoclonal antibody against the beta subunit showed that the alpha(1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca(2+) channel exhibits an asymmetric arrangement of auxiliary subunits.     

In vitro and in vivo phosphorylation of the Cav2.3 voltage-gated R-type calcium channel

During the recording of whole cell currents from stably transfected HEK-293 cells, the decline of currents carried by the recombinant human Cav2.3+β3 channel subunits is related to adenosine triphosphate (ATP) depletion after rupture of the cells. It reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials (Neumaier F., et al., 2018). Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. These findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. Protein phosphorylation is required for Ca_v2.3 channel function and could directly influence the normal features of current carried by these channels. Therefore, results from in vitro and in vivo phosphorylation of Ca_v2.3 are summarized to come closer to a functional analysis of structural variations in Ca_v2.3 splice variants.