Unmasking subtype-dependent susceptibility to C-type inactivation in mammalian Kv1 channels

Shaker potassium channels have been an essential model for studying inactivation of ion channels and shaped our earliest understanding of N-type vs. C-type mechanisms. In early work describing C-type inactivation, López-Barneo and colleagues systematically characterized numerous mutations of Shaker residue T449, demonstrating that this position was a key determinant of C-type inactivation rate. In most of the closely related mammalian Kv1 channels, however, a persistent enigma has been that residue identity at this position has relatively modest effects on the rate of inactivation in response to long depolarizations. In this study, we report alternative ways to measure or elicit conformational changes in the outer pore associated with C-type inactivation. Using a strategically substituted cysteine in the outer pore, we demonstrate that mutation of Kv1.2 V381 (equivalent to Shaker T449) or W366 (Shaker W434) markedly increases susceptibility to modification by extracellularly applied MTSET. Moreover, due to the cooperative nature of C-type inactivation, Kv1.2 assembly in heteromeric channels markedly inhibits MTSET modification of this substituted cysteine in neighboring subunits. The identity of Kv1.2 residue V381 also markedly influences function in conditions that bias channels toward C-type inactivation, namely when Na⁺ is substituted for K⁺ as the permeant ion or when channels are blocked by an N-type inactivation particle (such as Kvβ1.2). Overall, our findings illustrate that in mammalian Kv1 channels, the identity of the T449-equivalent residue can strongly influence function in certain experimental conditions, even while having modest effects on apparent inactivation during long depolarizations. These findings contribute to reconciling differences in experimental outcomes in many Kv1 channels vs. Shaker.