A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state

4-Aminopyridine Prevents the Conformational Changes Associated with P/C-Type Inactivation in Shaker Channels

ABSTRACT The effect of 4-aminopyridine (4-AP) on K v channel activation has been extensively investigated, but its interaction with inactivation is less well understood. Voltage-clamp fluorimetry was used to directly monitor the action of 4-AP on conformational changes associated with slow inactivation of Shaker channels. Tetramethylrhodamine-5-maleimide was used to fluorescently label substituted cysteine residues in the S3-S4 linker (A359C) and pore (S424C). Activation-and inactivation-induced changes in fluorophore microenvironment produced fast and slow phases of fluorescence that were modified by 4-AP. In Shaker A359C, 4-AP block reduced the slow-phase contribution from 61 Ϯ 3 to 28 Ϯ 5%, suggesting that binding inhibits the conformational changes associated with slow inactivation and increased the fast phase that reports channel activation from 39 Ϯ 3 to 72 Ϯ 5%. In addition, 4-AP enhanced both fast and slow phases of fluorescence return upon repolarization ( reduced from 87 Ϯ 15 to 40 Ϯ 1 ms and from 739 Ϯ 83 to 291 Ϯ 21 ms, respectively), suggesting that deactivation and recovery from inactivation were enhanced. In addition, the effect of 4-AP on the slow phase of fluorescence was dramatically reduced in channels with either reduced (T449V) or permanent P-type (W434F) inactivation. Interestingly, the slow phase of fluorescence return of W434F channels was enhanced by 4-AP, suggesting that 4-AP prevents the transition to C-type inactivation in these channels. These data directly demonstrate that 4-AP prevents slow inactivation of K v channels and that 4-AP can bind to P-type-inactivated channels and selectively inhibit the onset of C-type inactivation