The effects of S4 segments on the voltage-dependence of inactivation for Cav3.1 calcium channels

Abstract

T-type calcium channels exhibit fast voltage-dependent inactivation, for which the underlying structure-function relationship still remains unclear. To investigate the roles of S4 segments in voltage-dependent inactivation of T-type calcium channels, we created S4 replacement chimeras between Cav3.1 calcium channels (fast voltage-dependent inactivation) and Cav1.2 calcium channels (little voltage-dependent inactivation) by replacing S4s in Cav3.1 with the corresponding regions in Cav1.2. Wild type and chimeric channels were expressed in Xenopus oocytes and channel currents were recorded with two-electrode voltage-clamp. We showed that replacing S4 region in domain I shifted voltage-dependence for inactivation of Cav3.1 to the left, and the V 0.5 inact and k inact value were significantly changed. However replacing S4s in domains II-IV had no effects on the voltage-dependent inactivation properties. These results suggest that the roles of S4 segments in domains I-IV are different, and S4 in domain I is likely to be involved in voltage-dependent inactivation process. Its movement during membrane depolarization may trigger a conformational change in the inactivation gate.Cav3.1 calcium channel, S4 segment, voltage-dependent inactivation Voltage-dependent calcium channels play important roles in many cellular progresses, including second messenger cascades, neurotransmitter release, cardiac excitation and gene regulations. The molecular structure of the pore-forming subunit (α 1 ) of calcium channels has 4 homologous domains (I-IV), each with 6 transmembrane segments (S1-S6). To date, at least 10 genes have been cloned for the α 1 subunit. According to their electrophysiological characteristic, pharmacology and structure, these channels have been classified into 3 families: Cav1-Cav3. Cav3 family has markedly different biophysical characteristic from Cav1 family. Cav3 channels, displaying T-type current, are low-voltageactivating and have fast voltage-dependent inactivation. On the contrary, Cav1 channels, showing L-type current, are high-voltage-activating and have little voltage-dependent inactivation but having Ca 2+ -dependent inactivation instead [1][2][3] .Voltage-dependent ion channels form a huge family comprising potassium, sodium and calcium channels. For this family, S4 segments have similar conserved sequence. For sodium and potassium channels, it is well established that S4 segments play vital roles in the voltage sensing process. S4s rotate and move outward during membrane depolarization, and then induce channel activating and inactivating [4][5][6] . In contrast, there are very few studies carrying out on the roles of S4 segments in calcium channel. In our previous work, we started to investigate this issue by creating S4-replacement chimeras between Cav3.1 (α 1 G) channel (lowvoltage-activating and fast voltage-dependent inactivation) and Cav1.2 (α 1 C) (high-voltage-activating and little voltage-dependent inactivation) and also by using cysteine-scanning mutagenesis techniques. Our data