The “proline hinge”, a Proline-Valine-Proline (P-V-P) sequence, is a common structural motif in several voltage-gated ion channels. Kv1.4 is a voltage-gated K+ channel with a proline hinge at the intracellular side of S6. Kv1.4 (KCNA4), is a member of the Shaker-related family of voltage-gated K+ channels. Kv1.4 opens in response to depolarization, but then quickly inactivates to produce a transient outward current. This is thought to be the major molecular basis of Ito in the endocardial tissue of several mammalian species. In addition, this channel is thought to underlie an A type current in brain and smooth muscle. Kv1.4 channels have two distinct inactviation mechanisms: N- and C-type. N-type inactivation is rapid inactivation resulting from occlusion of the pore by the lipophilic N-terminal. C-type inactivation is slower, and involves structural changes on the intracellular and extracellular faces of the pore. We mutated the second proline to glycine or alanine: P558A and P558G. These mutations were studied in the presence and the absence of the N-terminal to separate the effects of the interaction between the proline hinge and N and C-type inactivation. Mutations were made in Kv1.4 and heterologously expressed in Xenopus oocytes as previously (1). All procedures were in accordance with the IACUC protocols and federal guidelines. Both the P558A and P558G mutations slowed or removed N- and C-type inactivation, as well as altering recovery from inactivation. P558A was very disruptive: activation was slowed more than an order of magnitude, and no inactivation was observed. The P558G mutation slowed N- and C-type inactivation by nearly an order of magnitude. The P558G mutation was sensitive to extracellular acidosis and intracellular quinidine binding, which suggests that transmembrane communication in N and C-type inactivation was preserved. Activation, studied in the absence of the N-terminal was also slowed and showed no sigmoid delay. Examination of the effects of the mutation using a modification of our previously reported Kv1.4 model (1) required a single rate limiting voltage insensitive step. However, the voltage dependence of activation required a positive shift in the voltage dependent steps of activation. A model with shifted voltage dependence of activation and a slow voltage insensitive preactivated to open transition accounted for the altered activation. This suggests that movement of S6 is energetically linked to early movements of the voltage sensor, as well as a final cooperative opening of the intracellular gate. Inactivation changes induced by the P558G required slowing of N- and C-type inactivation. These findings are consistent with significant structural rearrangements involving the intracellular side of S6 during C-type inactivation and our hypothesis that the proline hinge plays a significant role in inactivation and recovery.