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.
Highlights
Timothy syndrome (TS),3 an autosomal dominant disorder, arises from two de novo missense mutations, G402S and G406R, in CaV1.2 calcium channels
We have previously shown that mutations in the lower third of S6 segments in CaV1.2 in most cases shift the channel inactivation and activation in a coupled manner
Do mutations of the homologous small residues in the other domains, Ala-780(IIS6), Gly-1193(IIIS6), and Ala-1503(IVS6), have similar effects on channel gating to substitutions of Gly432? Second, how do amino acid substitutions in these positions affect the link between activation and inactivation? Third, is an essential role of the G/A/G/A residues in helix packing and closed-state stability supported by functional data? Answers to these questions will help to understand the interactions of residues in the bundle crossing region and help to clarify the specific impact of pore residues on activation and inactivation
Summary
Timothy syndrome (TS), an autosomal dominant disorder, arises from two de novo missense mutations, G402S and G406R, in CaV1.2 calcium channels. Functional studies have shown that TS mutations G402S and G406R dramatically reduce voltage-dependent channel inactivation, resulting in sustained membrane depolarization and increased calcium entry during an action potential (1). We have previously shown that mutations in the lower third of S6 segments in CaV1.2 in most cases shift the channel inactivation and activation in a coupled manner. The functional impact and structural basis of amino acid substitutions of the TS Gly-432 (corresponding to Timothy Gly-402 in human CaV1.2) are less understood. Our homology model suggests that Gly-432 forms part of a conserved group of small amino acids, Gly-432(IS6), Ala-780(IIS6), Gly1193(IIIS6), and Ala-1503(IVS6), near the inner channel mouth of CaV1.2 (Fig. 1), which we call the G/A/G/A motif. Do mutations of the homologous small residues in the other domains, Ala-780(IIS6), Gly-1193(IIIS6), and Ala-1503(IVS6), have similar effects on channel gating to substitutions of Gly432? Do mutations of the homologous small residues in the other domains, Ala-780(IIS6), Gly-1193(IIIS6), and Ala-1503(IVS6), have similar effects on channel gating to substitutions of Gly432? Second, how do amino acid substitutions in these positions affect the link between activation and inactivation? Third, is an essential role of the G/A/G/A residues in helix packing and closed-state stability supported by functional data? Answers to these questions will help to understand the interactions of residues in the bundle crossing region and help to clarify the specific impact of pore residues on activation and inactivation
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