Channelopathies in Cav1.1, Cav1.3, and Cav1.4 voltage-gated L-type Ca2+ channels.
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ABSTRACT:
SUBMITTER: Striessnig J
PROVIDER: S-EPMC2883925 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cav1.4 L-type Ca(2+) channels are crucial for synaptic transmission in retinal photoreceptors and bipolar neurons. Recent studies suggest that the activity of this channel is regulated by the Ca(2+)-binding protein 4 (CaBP4). In the present study, we explored this issue by examining functional effects of CaBP4 on heterologously expressed Cav1.4. We show that CaBP4 dramatically increases Cav1.4 channel availability. This effect crucially depends on the presence of the C-terminal ICDI (inhibitor of Ca(2+)-dependent inactivation) domain of Cav1.4 and is absent in a Cav1.4 mutant lacking the ICDI. Using FRET experiments, we demonstrate that CaBP4 interacts with the IQ motif of Cav1.4 and that it interferes with the binding of the ICDI domain. Based on these findings, we suggest that CaBP4 increases Cav1.4 channel availability by relieving the inhibitory effects of the ICDI domain on voltage-dependent Cav1.4 channel gating. We also functionally characterized two CaBP4 mutants that are associated with a congenital variant of human night blindness and other closely related nonstationary retinal diseases. Although both mutants interact with Cav1.4 channels, the functional effects of CaBP4 mutants are only partially preserved, leading to a reduction of Cav1.4 channel availability and loss of function. In conclusion, our study sheds new light on the functional interaction between CaBP4 and Cav1.4. Moreover, it provides insights into the mechanism by which CaBP4 mutants lead to loss of Cav1.4 function and to retinal disease.
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Project description:Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (I(Na)) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named "channel partners" or "channel interacting proteins" (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium "channelopathies". This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field.
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Project description:Ca(2+) influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated CaV1.3 Ca(2+) channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca(2+) imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca(2+) channels, resulting in reduced synaptic Ca(2+) influx. Using superresolution microscopy, we found that Ca(2+) channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca(2+) current, whereas the apparent Ca(2+) dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca(2+) influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca(2+) channels at IHC AZs and are required for normal hearing.
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Project description:Integration of voltage-gated Ca(2+) channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca(2+) channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the ?1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca(2+) channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba(2+) current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca(2+) channel activity in defined synaptic membrane compartments and thereby influences transmitter release.
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Project description:Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entryways for Ca(2+) in excitable cells are high-voltage activated (HVA) Ca(2+) channels. These are plasma membrane proteins composed of several subunits, including α(1), α(2)δ, β, and γ. Although the principal α(1) subunit (Ca(v)α(1)) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Ca(v)β) plays an essential role in regulating the surface expression and gating properties of HVA Ca(2+) channels. Ca(v)β is also crucial for the modulation of HVA Ca(2+) channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca(2+) channels by binding to Ca(v)β. There are also indications that Ca(v)β may carry out Ca(2+) channel-independent functions, including directly regulating gene transcription. All four subtypes of Ca(v)β, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Ca(v)βs reveal how they interact with Ca(v)α(1), open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Ca(v)β, with both a historical perspective as well as an emphasis on recent advances.
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Project description:Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III-IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.
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Project description:Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects.
| S-EPMC2785611 | biostudies-literature
Project description:Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 α1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand-like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.
| S-EPMC5749111 | biostudies-literature
Project description:Cav1.3 L-type Ca(2+) channel is known to be highly expressed in neurons and neuroendocrine cells. However, we have previously demonstrated that the Cav1.3 channel is also expressed in atria and pacemaking cells in the heart. The significance of the tissue-specific expression of the channel is underpinned by our previous demonstration of atrial fibrillation in a Cav1.3 null mutant mouse model. Indeed, a recent study has confirmed the critical roles of Cav1.3 in the human heart (Baig, S. M., Koschak, A., Lieb, A., Gebhart, M., Dafinger, C., Nürnberg, G., Ali, A., Ahmad, I., Sinnegger-Brauns, M. J., Brandt, N., Engel, J., Mangoni, M. E., Farooq, M., Khan, H. U., Nürnberg, P., Striessnig, J., and Bolz, H. J. (2011) Nat. Neurosci. 14, 77-84). These studies suggest that detailed knowledge of Cav1.3 may have broad therapeutic ramifications in the treatment of cardiac arrhythmias. Here, we tested the hypothesis that there is a functional cross-talk between the Cav1.3 channel and a small conductance Ca(2+)-activated K(+) channel (SK2), which we have documented to be highly expressed in human and mouse atrial myocytes. Specifically, we tested the hypothesis that the C terminus of Cav1.3 may translocate to the nucleus where it functions as a transcriptional factor. Here, we reported for the first time that the C terminus of Cav1.3 translocates to the nucleus where it functions as a transcriptional regulator to modulate the function of Ca(2+)-activated K(+) channels in atrial myocytes. Nuclear translocation of the C-terminal domain of Cav1.3 is directly regulated by intracellular Ca(2+). Utilizing a Cav1.3 null mutant mouse model, we demonstrate that ablation of Cav1.3 results in a decrease in the protein expression of myosin light chain 2, which interacts and increases the membrane localization of SK2 channels.
| S-EPMC4335206 | biostudies-literature
Project description:Voltage-gated Ca(2+) (Cav) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Cav channels are subject to diverse forms of regulation, including those involving the Ca(2+) ions that permeate the pore. High voltage-activated Cav channels undergo Ca(2+)-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Cav1 (L-type) and Cav2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Cav channel. HEK-293T cells do not express endogenous Cav channels, but Cav channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca(2+)-dependent modulation of recombinant Cav channels in HEK-293T cells.
| S-EPMC5162803 | biostudies-literature