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      Proximal clustering between BK and Ca V1.3 channels promotes functional coupling and BK channel activation at low voltage

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          Abstract

          Ca V-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and Ca V1.3 channels, unique Ca V1 channels that activate at low voltages. We found that when BK and Ca V1.3 channels were co-expressed in the same cell, BK channels started activating near −50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated Ca V2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of Ca V1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of Ca V1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.

          DOI: http://dx.doi.org/10.7554/eLife.28029.001

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          Most cited references51

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          Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels.

          Voltage-gated L-type Ca2+ channels (LTCCs) containing a pore-forming alpha1D subunit (D-LTCCs) are expressed in neurons and neuroendocrine cells. Their relative contribution to total L-type Ca2+ currents and their physiological role and significance as a drug target remain unknown. Therefore, we generated D-LTCC deficient mice (alpha1D-/-) that were viable with no major disturbances of glucose metabolism. alpha1D-/-mice were deaf due to the complete absence of L-type currents in cochlear inner hair cells and degeneration of outer and inner hair cells. In wild-type controls, D-LTCC-mediated currents showed low activation thresholds and slow inactivation kinetics. Electrocardiogram recordings revealed sinoatrial node dysfunction (bradycardia and arrhythmia) in alpha1D-/- mice. We conclude that alpha1D can form LTCCs with negative activation thresholds essential for normal auditory function and control of cardiac pacemaker activity.
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            Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits

            To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.
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              Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity.

              The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for alpha(1D) (Ca(v)1.3) Ca(2+) channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (I(Ca,L)) activating at negative voltages. Pharmacological analysis of I(Ca,L) demonstrates that Ca(v)1.3 gene inactivation specifically abolishes I(Ca,L) in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Ca(v)1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                30 June 2017
                2017
                : 6
                : e28029
                Affiliations
                [1 ]deptDepartment of Physiology and Biophysics , University of Washington , Seattle, United States
                [2 ]deptDepartment of Physiology and Membrane Biology , University of California, Davis , Davis, United States
                National Institutes of Health , United States
                National Institutes of Health , United States
                Author notes
                [†]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-0964-385X
                http://orcid.org/0000-0002-4297-8029
                Article
                28029
                10.7554/eLife.28029
                5503510
                28665272
                c9944a8a-9f67-4243-83b3-86f06cf38538
                © 2017, Vivas et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 23 April 2017
                : 28 June 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;
                Award ID: R37NS008174
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000050, National Heart, Lung, and Blood Institute;
                Award ID: R01HL085686
                Award Recipient :
                Funded by: Wayne E. Crill Endowed Professorship;
                Award ID: Professor Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000050, National Heart, Lung, and Blood Institute;
                Award ID: R01HL085870
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biophysics and Structural Biology
                Custom metadata
                2.5
                Super-resolution imaging reveals a novel multi-channel arrangement that explains how big potassium (BK) channel activation closely tracks the opening of low-voltage-activated L-type calcium channels.
                2.5

                Life sciences
                bk channels,voltage-gated calcium channels,sympathetic neurons,hippocampal neurons,super-resolution microscopy,ground-state depletion,rat

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