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

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC, Canada.
The Journal of General Physiology (Impact Factor: 4.79). 06/2007; 129(5):437-55. DOI: 10.1085/jgp.200709774
Source: PubMed


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 alpha-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.

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    • "Although P/C-type inactivation is classically viewed as an open channel inactivation mechanism (see Figures 1A and 2C), recent work has shown that the P-gate may also undergo inactivation when the channel is still closed. Claydon et al. (2007, 2008) have performed electrophysiological measurements combined with voltage-clamp fluorimetry to examine whether ShakerIR (N-type inactivation removed) channels may undergo P/C-type inactivation also from closed-states. Acidic pH, which promotes rearrangements at the P-gate, and the Shaker ILT triple-mutant (Smith-Maxwell et al., 1998), which segregates channel opening from voltage-dependent activation by shifting the respective curves apart, were exploited in this study. "
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    • "It should be noted that sensing currents only capture movement of charges in the direction of the membrane electric field, whereas the fluorescence signal can report additional conformational transitions either directly reflecting movement of charged residues or conformational transitions that are triggered by the former. In this sense, the fluorescence signal of VSFP2.3 contains information similar to that obtained by voltage-clamp fluorometry (Claydon et al. 2007; Pathak et al. 2007; Villalba-Galea 2008a, b). For Ci-VSP it has been shown that the activated position of its VSD is not stable, thus causing the VSD to relax into a lower energy conformational state at prolonged depolarizations (Villalba-Galea et al. 2008b). "
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    • "For example, as with slow inactivation in ShIR (L ´ opez-Barneo et al. 1993; Baukrowitz & Yellen, 1995; Kiss & Korn, 1998), the resting inactivation induced by low pH is antagonized by elevated [K + ] o (K d = 1 mM; Jäger & Grissmer, 2001; Kehl et al. 2002). Additionally, mutation of a specific outer pore residue (Kv1.5 R487 or ShIR T449) to a valine or tyrosine, markedly attenuates the enhanced current decay and resting inactivation observed at low pH (L ´ opez-Barneo et al. 1993; Kehl et al. 2002; Trapani & Korn, 2003; Starkus et al. 2003; Claydon et al. 2007). Although in ShIR the site of action for the enhanced slow inactivation observed at low pH has been suggested to be a conserved aspartate residue in the GYGD sequence of the selectivity filter (Pérez-Cornejo, 1999; Starkus et al. 2003), in Kv1.5 the pH sensitivity is dramatically reduced by the mutation to glutamine of the H463 residue in the turret of each α subunit (Steidl & Yool, 1999; Kehl et al. 2002). "
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