The Role of Intracellular Domain of S6 during Kv1.4 Inactivation
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Voltage-gated potassium (Kv) channels are a large and diverse class of transmembrane ion channels that are selective for K + over other cations. A typical voltage-gated potassium channel is composed of four identical &agr; subunits which have six transmembrane segments (S1-S6). S5 and S6 from each subunit line up around the central axis of the tetramer to form the pore domain which is surrounded by S1-S4 forming the voltage sensing domain. In response to a depolarizing voltage, the movement of S4 transduced to S6, resulting in an open and conductive channel. Under a sustained stimulus, the channels enter into a non-conductive state called inactivation. In order to investigate the role of S6 during gating, the Kv1.4 channel (encoded by KCNA4 in human), which is a member of Shaker -related Kv channel family, was examined in this study. Kv1.4 is inactivated by two different mechanisms: N-type and C-type inactivation. N-type inactivation is the occlusion of the pore with the N-terminal "ball", whereas C-type inactivation involves a more generalized movement of the pore, involving both the intracellular and extracellular sides of the channel. Because the "prolinehinge", a Proline-Valine-Proline (P-V-P) sequence, provides the flexibility for movement of S6 and is important for channel opening, inactivation and their coupling, we mutated the second proline in the proline hinge. We showed that after mutation, N- and C-type inactivation had parallel shifts in steady-state inactivation which mirror the changes in activation. The reduction in the rate of activation also slowed or inhibited the both N- and C-type inactivation. Since proline hinge form a "kink" in S6 and is critical for inactivation, it likely breaks S6 into two discrete blocks. To study the sequence of conformational changes of extracellular pore region and intracellular S6 domain, we applied &phis; value analysis to these two domains which is thought important for C-type inactivation. Previous studies indicated that V561 might be the potential intracellular activation gate and mutation at an equivalent position in Kv1.1courses episodic ataxia. In our study, V561 was mutated to a series of other amino acids to perturb the free energy of the channel. We found that the conformational change of intracellular S6 occurs early in N-type inactivation, and meanwhile it occurs before the conformational change of the extracellular pore region during C-type inactivation.