Functions of the Src homology 3 and guanylate kinase domains of the [beta]-subunit of voltage-gated calcium channels

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Mutations of Nonconserved Residues within the Calcium Channel α1-interaction Domain Inhibit β-Subunit Potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (CaVα1) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (CaVβ) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. CaVβ binds reversibly to a conserved consensus sequence in CaVα1, the α1-interaction domain (AID), which forms an α-helix when complexed with CaVβ. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on CaVβ. Here, we studied the effect of mutating residues located opposite to the AID-CaVβ contact surface in CaV1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other CaVα1 subunits (E462R, K465N, D469S, and Q473K) hinders CaVβ's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for CaVβ. At the single channel level, these CaV1.2 mutants coexpressed with CaVβ2a visit high open probability mode less frequently than wild-type channels. On the other hand, CaV1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs CaVβ binding), or a deletion of the whole AID sequence, does not exhibit CaVβ-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of CaVβ, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that CaVβ-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that CaVβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by CaVβ

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl− currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected

Ca V

Membrane depolarization activates the multisubunit CaV1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV1.2-mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV1.2α1 pore-forming and auxiliary CaVβ subunits and analyzed their trafficking dynamics from single-particle-tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest-mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaVβ to CaV1.2α1 suggesting that high-speed transport occurs in association with CaVβ. Selective tracking of CaV1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaVβ-independent retrograde transport. Retrograde speeds of CaV1.2α1 are compatible with myosin VI-mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV1.2α1 was covalently linked to CaVβ. Thus, CaVβ promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L-type calcium channels

Ca V β controls the endocytic turnover of Ca V 1 .2 L‐type calcium channel

Membrane depolarization activates the multisubunit CaV1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV1.2-mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV1.2α1 pore-forming and auxiliary CaVβ subunits and analyzed their trafficking dynamics from single-particle-tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest-mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaVβ to CaV1.2α1 suggesting that high-speed transport occurs in association with CaVβ. Selective tracking of CaV1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaVβ-independent retrograde transport. Retrograde speeds of CaV1.2α1 are compatible with myosin VI-mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV1.2α1 was covalently linked to CaVβ. Thus, CaVβ promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L-type calcium channels

Single channel mean currents and I/Q plots from macroscopic currents from different Ca1

2 variants recorded in high Ba and S(-)Bay K8644. (A) Mean current traces for six patches containing single Ca1.2 WT/Caβ channels (black) from seven patches with Ca1.2 E462R/Caβ channels (blue), and from 6 with Ca1.2 K465N/Caβ channels (red). The number of traces averaged in each case was 4,032 for Ca1.2 WT/Caβ, 7,504 for Ca1.2 E462R/Caβ, and 6,104 for Ca1.2 K465N/Caβ. Voltage protocol and recording condition were as described in . Calibration bars correspond to 50 ms and 100 fA. (B) I/Q versus voltage plot for Ca1.2 WT ( = 12), Ca1.2 E462R ( = 12), and Ca1.2 K465N ( = 13) coexpressed with Caβ and recorded in external 76 mM Ba and 0.1 μM of S(-) Bay K 8644 as used for single channel.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Deletion of AID sequence or replacement of the conserved tryptophan abolished modulation by Caβ

(A) Superimposed macroscopic current traces from oocytes coexpressing Caβ either with Ca1.2 W470S or Ca1.2 ΔAID. Each trace was obtained during a 70-ms pulse of increasing amplitude, starting at −40 mV and ending at 150 mV in 10-mV increments. Membrane was held at −80 mV until the beginning of the pulse and returned to −40 mV for the remaining of the trace (shown at the top). Currents were sampled at 2.5 kHz until 3 ms before the end of the pulse, and then at 50 kHz. Traces were filtered at 10 kHz, and a P/−4 prepulse protocol was used to subtract linear components. Calibration bars correspond to 20 ms and 200 nA. (B) Conductance–voltage relationship (GV curve) for the different subunit combinations shown in A. The peak amplitude of the tail currents for each test voltage was normalized by the largest tail current (I/Imax) to generate the GV curves. Open and filled symbols correspond to oocytes recorded with or without Caβ, respectively. The sums of two Boltzmann distributions that best described each set of data are shown as continuous lines.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation-4

I/Q versus voltage from oocytes expressing Ca1.2 E462R after the injection of purified Caβ protein at the indicated concentrations. Traces correspond to superimposed responses to three 60-ms depolarizing pulses to −30 mV, 0 mV, and +30 mV from a holding voltage of −90 mV. Calibration bars correspond to 20 ms and 200 nA. Experimental I/Q values (8) were fitted to the equation (blue line):Each member of the equation corresponds to templates in absence (−) or presence (+) of saturating concentration of Caβ protein (2.0 μM). Variables defining each template were obtained from the fit to average I/Q plot from each condition. The contribution of +β and −β templates are shown as green and red lines, respectively. (B) As A) but for Ca1.2 K465N. (C) Mean ± SE of β2a-like versus protein concentration ([Caβ]) in μM. Continuous lines show the fit to a standard Hill equation:Where is the apparent dissociation constant and is the Hill coefficient. ranged between 1.4 and 1.6, whereas for WT, E462R, and K465N was 0.20, 0.22, and 0.25 μM, respectively. The number of averaged experiments ranged from three to six for every concentration and calcium channel variant.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p