Activation and Inactivation Steps Altered by Proline Hinge Mutations in Kv1.4

Abstract

Several voltage-gated channels share a Proline-Valine-Proline (“proline hinge”) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine (P558G) or alanine (P558A), and studied these mutations in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N and C-type inactivation.Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that the transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement plays a significant role in inactivation and recovery.To analyze the effect of these mutations in changing channel gating kinetics, we modified our previously published model of Kv1.4 by adding a primed but non-conducting state connected to the open state by a voltage insensitive step. In order to reproduce the experimental observation that N-type inactivation occurs at more positive potentials than C-type, the C-type inactivated state must be coupled to earlier voltage-dependent steps. Analyses of this model suggest that both P558G and P558A mutations not only modified early voltage dependent steps, but also made the voltage insensitive transition from the primed state to the open state very slow relative to activation.