A Proline Scan Approach to Investigate the Activation Gate of hERG Channels

Abstract

In Shaker channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline (PVP) motif, which introduces a kink in the helices that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go related gene (hERG) channels lack the PVP motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we performed a proline scan of the inner S6 helix, from I655 to Y667, to determine the position of the gate. The rationale was that proline-induced S6 disruption would impede gate function when a proline was engineered above, but not below, the native gate region. We discovered that proximal substitutions (I655P-Q664P) impeded gate closure trapping channels in the open state, while distal substitutions (R665P, L666P and Y667P) preserved wild type-like gating. That proline substitutions below Q664 preserved channel gating, while residues above disrupted gate function, strongly suggests that the position of the intracellular gate is formed at Q664. These data are consistent with previous homology model-based predictions1. Interestingly, in V659P channels the gate was trapped open, but upon strong hyperpolarization channels slowly activated into a distinct voltage-dependent open state, reminiscent of the well-studied hERG mutation, D540K. The presence of voltage-dependent gating in this mutant suggests that the trapped open phenotype is due to uncoupling of gate closure from voltage sensor gating, rather than an immobilization of sensor movement. Moreover, the activation of V659P channels upon hyperpolarization suggests a ‘down' configuration of the voltage sensor that is distinct from that occupied at −80 mV and that leads to hyperpolarization-activated pore opening, as in HCN channels.1 Wynia-Smith et. al., J. Gen. Physiol. 132:507-520, 2008.