Timothy Mutation Disrupts the Link between Activation and Inactivation in CaV1.2 Protein

Abstract

The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of CaV1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for CaV1.2 activation and inactivation gating are discussed.

Hyperactivation of L-type voltage-gated Ca2+ channels in C. elegans striated muscle can result from point mutations in the IS6 or the IIIS4 segment of the α1 subunit.

“…The n2368 mutation replaces the second glycine by an arginine. Such a change has not been described before; however, the effects of replacement of this glycine by methionine, asparagine, alanine, serine or tryptophane have been analysed in rabbit L-type channels expressed in heterologous systems (Depil et al, 2011). Depending on the mutations, the half-activation potential was shifted towards more negative potentials, as we observed for egl-19(n2368), or towards more positive ones.…”

“…In particular, glycine residues of the GX 9 GX 3 G motif are involved in activation and inactivation mechanisms: mutations of these residues lead to a shift of the half-activation potential and a decrease of inactivation speed (Splawski et al, 2004;Splawski et al, 2005;Raybaud et al, 2006;Barrett and Tsien, 2008;Cens et al, 2008;Depil et al, 2011;Yazawa et al, 2011). Mutations of the second and third glycine have been identified in patients suffering from Timothy syndrome, a multisystem disorder mainly characterized by cardiac arrhythmias, autism and syndactyly (Splawski et al, 2004;Splawski et al, 2005).…”

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

“…Structurally, the glycine residue at 402 position of IS6, together with the same positions in the IIS6, IIIS6 and IVS6 segments, form the G/A/G/A motif, which is near the inner channel mouth of Ca V 1.2. These four residues interact with larger bulky residue from neighboring S6 helices to stabilize the inactivation gate [68]. In TS2, in addition to p.G406R mutation, p.G402S will also introduce a more bulky serine residue to disrupt the tightly sealing of the S6 helices, resulting in the slower deactivation [68].…”

“…These four residues interact with larger bulky residue from neighboring S6 helices to stabilize the inactivation gate [68]. In TS2, in addition to p.G406R mutation, p.G402S will also introduce a more bulky serine residue to disrupt the tightly sealing of the S6 helices, resulting in the slower deactivation [68]. Although neither heterozygous nor homozygous p.G402S transgenic mice can survive to weaning because of the high expression of exon 8 and a fatal extremely high level of calcium channels mutation, TS2-like mouse model with p.G406R had been generated by heterozygously keeping an inverted neomycin cassette in exon 8A [79], these mice could survive through adulthood due to the lowered expression of p.G406R LTCCs.…”

An Update on Channelopathies

“…Failure of the calcium channel to close properly may be responsible for after-depolarizations during repolarization and induce long-QT arrhythmias. 27 We now return briefly to the fascinating proteins that behave as channelopathies but are not strictly channel forming. LQT4 is caused by mutation of a membrane adaptor protein, ankyrin-B, at transverse-tubule and sarcoplasmic reticulum sites.…”