Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels

Abstract

Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30–40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.

Increase of CaV3 channel activity induced by HVA β1b-subunit is not mediated by a physical interaction

“…Whole-cell Ca 2+ (and Na + ) currents were recorded at room temperature (21–23 °C) with the patch-clamp technique following the methods of Sanchez-Sandoval et al [ 35 ]. External solutions composition was as follows (in mM): for LVA channels, 5 CaCl 2 and 175 TEA-Cl; for HVA channels, 10 BaCl 2 and 152 TEA-Cl; and for sodium channels, 158 NaCl, 2 CaCl 2 , and 2 MgCl 2 .…”

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

“…Detailed studies on domain I by the same group pointed out its importance for channel activation and showed that the S5 and S6 region rather than voltage sensing S1-S4 were critical [9]. Swapping of S4 segments between Cav3.3 channels and Ca V 1.2 channels demonstrated that IIS4 and, to a lesser degree IVS4, segments are crucial in determining the negative voltage threshold of Ca V 3.3 channel activation [10]. …”