Key result
In the presence of 20 µM Zn2+, the activation currents of L366H:F370H mutant Shaker channels were slowed down by a factor of 3.5 and the voltage-dependence shifted by 10 mV towards depolarized potentials, indicating stabilization of an α-helical conformation in a closed state.
Why the study?
Whether a helix transition from alpha- to 3 10-helix occurs in the S4 segment during voltage-gated ion channel activation remained unresolved.
Effect estimate: slowed down by a factor of 3.5, 10 mV shift
Coordination of engineered histidine residues by Zn2+ stabilizes the α-helical conformation of the S4 segment in Shaker potassium channels, suggesting a transient secondary structure change during activation.
Does not alter arrhythmia management; leaves open whether S4 helix stabilization modulates native cardiac Kv channel gating.
Voltage-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 3 10 -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, in i, i+3 and i, i+6 or i, i+4 and i, i+7 pairs, to favor 3 10 - or α-helical conformations, respectively. We have taken advantage that His can be coordinated by Zn +2 to 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 the active state in the activation process. STATEMENT OF SIGNIFICANCE Conformational transitions between α-helix and 3 10 -helix in the S4 segment of Shaker potassium channel during gating has been under debate. The present study shows the coordination by Zn 2+ 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 +2 the 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 the resting to the active state during activation.
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Bassetto et al. (2019) studied this question. L366H:F370H mutation with 20 µM Zn2+ vs. Absence of Zn2+ or wild-type channels was evaluated on Activation current kinetics and voltage-dependence (slowed down by a factor of 3.5, 10 mV shift). In the presence of 20 µM Zn2+, the activation currents of L366H:F370H mutant Shaker channels were slowed down by a factor of 3.5 and the voltage-dependence shifted by 10 mV towards depolarized potentials, indicating stabilization of an α-helical conformation in a closed state.
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