Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel’s SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., T T VGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the T T VGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K + concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1.

Voltage-gated K+ (Kv) channels mediate the flow of K+ across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K+ over Na+ selectivity, and even NMDG+ can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation.

Substituting the pore residue W434 in the Shaker Kv channel by a phenylalanine (W434F) accelerates its C-type inactivation, which involves the collapse of the selectivity filter (SF). In Shaker-W434F this collapse precedes S6-gate opening upon activation and no ionic currents are recorded. However, a valine mutation for the residue T449 (T449V) slows inactivation and introducing this T449V substitution in the Shaker-W434F rescues ionic conduction. The human Shaker-type Kv1.2 channel has a valine at the homologous T449 position (V381). Interestingly, the Kv1.2-W366F mutant yielded ionic current (similar to the Shaker-W434F-T449V combination). Accordingly, substituting V381 in Kv1.2-W366F by a threonine resulted in a similar (non-conducting) phenotype as Shaker-W434F. Thus the rate of SF collapse can be controlled by specific combinations of pore residue mutations. Patch-clamp analysis of the ionic currents of Kv1.2-W366F and Shaker-W434F-T449V showed that the channels remained K þ selective. Thus, the SF of Shaker-W434F-T449V adopts a K þ-selective conformation when the S6-gate opens. To study the SF conformation of Shaker-W434F when the S6-gate closes, we combined Shaker-W434F with the mutation P475D, that by itself prevents complete closing of the S6-gate. We expected to record ionic currents of the double Shaker-W434F-P475D mutant at hyperpolarized potentials when the S6-gate closes and the SF recovers from its collapsed inactivated state. Indeed, Shaker-W434F-P475D resulted in functional voltagedependent channels that were conducting at hyperpolarized potentials and ceased conducting at depolarized potentials. Interestingly, this conductive state of Shaker-W434F-P475D was both Na þ and K þ permeable, i.e. the high K þ selectivity was lost. The mutant remained, however, sensitive to external TEA block. Thus, preventing full closure of the S6-gate appears to affect the recovery process of the SF such that it is trapped in a conformation conducting both Na þ and K þ .

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