The effect of the outermost basic residues in the S4 segments of the CaV3.1 T-type calcium channel on channel gating

Abstract

The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca(V)3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca(V)3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it.

Role of individual S4 segments in gating of Cav3.1 T-type calcium channel by voltage

“…The most prominent effect on channel conductance was caused by single mutation in domain III or by double mutation in domains I and II. Qualitatively similar changes were reported previously [12]. Different compositions of bath and pipette solutions do not allow us to compare previously reported values to those from our current study.…”

“…cDNA for the channel was cloned into pEGFP N-1 plasmid (Clontech, Mountain View, CA, USA) with the stop codon removed, so that EGFP protein was fused to a carboxy terminus of the channel protein and enabled visual detection of successfully transfected cells. Mutations were introduced into S4 segments in individual channel domains using PCR-based methods [12]. Uppermost arginines in individual S4 segments were replaced by cysteines (R180C, R834C, R1379C, and R1717C).…”

“…Series of four single mutants with a replacement in the single domain, four double mutants with replacements in two neighboring domains, and one quadruple mutant with replacement in all four channel domains were constructed. Detailed procedures were described previously [12]. …”

“…Effect of substitution of uppermost charged arginines by neutral cysteines in individual domains of the Ca V 3.1 channel showed the role of S4 segments in domains I, II and III in channel activation [12]. The importance of the domain I in Ca V 3 channel gating was underscored by several studies by the Perez-Reyes group describing a role of the putative gating brake in the intracellular loop connecting domains I and II in stabilizing the closed state of the channel [13,14].…”

Cysteines in the loop between IS5 and the pore helix of CaV3.1 are essential for channel gating

“…Similarly, swapping parts of Ca V 3.1 and Ca V 3.3 channels pointed to a central role of domain IV and to a lesser impact of domain I [8]. Exchange of the basic uppermost arginines in each of the S4 segments by neutral cysteines substantiated the importance of the IIIS4 segment and a moderate role of the segments IS4 and IIS4 [17].…”