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Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation.

ABSTRACT: Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to -26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and -4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± 0.7 mV and IVS4(K5Q): 8.1 ± 0.7 mV vs WT: 14.1 ± 0.8 mV). IS4(K1Q) attenuated while IS4(K1Q/R2Q/R3Q) and IS4(K1Q/R2Q/R4Q/R3Q) enhanced the development of inactivation. Shifts in the voltage dependence of inactivation curves induced by IS4 neutralizations significantly correlated with shifts of the voltage dependence of channel activation (r = 0.95, p < 0.01) indicating that IS4 movement is not only rate limiting for activation but also initiates inactivation. The paradoxical decrease of the slope factor of the steady-state inactivation and acceleration of inactivation kinetics upon charge neutralization in segment IS4 may reflect the loss of stabilizing interactions of arginines and lysine with surrounding residues.

SUBMITTER: Andranovits S

PROVIDER: S-EPMC5629230 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation.

Andranovits Stanislav S Beyl Stanislav S Hohaus Annette A Zangerl-Plessl Eva Maria EM Timin Eugen E Hering Steffen S

Pflugers Archiv : European journal of physiology 20170801 11

Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to -26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of ...[more]

PMID: 28766141

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Project description:The CaV1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its ?1C pore-forming subunit is known to undergo extensive alternative splicing to produce many CaV1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human CaV1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with CaV?2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V1/2inact of CaV1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V1/2act. However, the measured effects were ?-subunit-dependent when comparing CaV?2a with CaV?3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing CaV1.2 as compared to exon-1a-containing CaV1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with CaV?2a or CaV?3 subunit. This finding correlated well with a higher channel surface expression for the exon 1-CaV1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human CaV1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.

Dataset Information

Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation.

Publications

Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation.

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