Metal bridge in S4 segment supports helix transition inShakerchannel

Abstract

ABSTRACTVoltage-gated ion channels play important roles in physiological processes, especially in excitable cells, where they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared: the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, possible helix transition from α-to 310-helix in S4 during activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, ini, i+3andi, i+6ori, i+4andi, i+7pairs, to favor 310- or α-helical conformations, respectively. We have taken advantage that His can be coordinated by Zn+2to promote metal ion bridges and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in α-helical conformation). In presence of 20 μM of Zn+2, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, the voltage-dependence is shifted by 10 mV towards depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in α-helical conformation a closed(resting or intermediate)state is stabilized rather than destabilizing the open (active)state. Taken together, our data indicates that the S4 undergoes α-helical conformation to a short-lived different secondary structure transiently before reaching theactivestate in the activation process.STATEMENT OF SIGNIFICANCEConformational transitions between α-helix and 310-helix in the S4 segment ofShakerpotassium channel during gating has been under debate. The present study shows the coordination by Zn2+of a pair of engineered histidine residues (L366H:F370H) in the intermediate region of S4 in Shaker, favoring α-helical conformation. In presence of 20μM of Zn+2the activation currents of L366H:F370H channels become slower, with 10 mV positive shift in the voltage-dependence and no effects on deactivation time constants suggesting a stabilization of a closed state rather than destabilization the open(active)state. Collectively, our data indicate that S4 undergoes secondary structure changes, including a short-lived secondary structure transition, when S4 moves from therestingto theactivestate during activation.