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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Available from: David Fedida, Oct 04, 2015
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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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    ABSTRACT: In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive ("P/C-type inactivation"). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as "preferential" closed-state inactivation, we introduce the term "A/C-type inactivation". Mechanistically, P/C- and A/C-type inactivation represent two forms of "voltage sensor inactivation."
    Frontiers in Pharmacology 05/2012; 3:100. DOI:10.3389/fphar.2012.00100 · 3.80 Impact Factor
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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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    ABSTRACT: A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.
    Biophysics of Structure and Mechanism 11/2010; 39(12):1625-35. DOI:10.1007/s00249-010-0620-0 · 2.22 Impact Factor
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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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    ABSTRACT: External H+ and Ni2+ ions inhibit Kv1.5 channels by increasing current decay during a depolarizing pulse and reducing the maximal conductance. Although the former may be attributed to an enhancement of slow inactivation occurring from the open state, the latter cannot. Instead, we propose that the loss of conductance is due to the induction, by H+ or Ni2+, of a resting inactivation process. To assess whether the two inactivation processes are mechanistically related, we examined the time courses for the onset of and recovery from H+- or Ni2+-enhanced slow inactivation and resting inactivation. Compared to the time course of H+- or Ni2+-enhanced slow inactivation at +50 mV, the onset of resting inactivation induced at 80 mV with either ion involves a relatively slower process. Recovery from slow inactivation under control conditions was bi-exponential, indicative of at least two inactivated states. Recovery following H+- or Ni2+-enhanced slow inactivation or resting inactivation had time constants similar to those for recovery from control slow inactivation, although H+ and Ni2+ biased inactivation towards states from which recovery was fast and slow, respectively. The shared time constants suggest that the H+- and Ni2+-enhanced slow inactivated and induced resting inactivated states are similar to those visited during control slow inactivation at pH 7.4. We conclude that in Kv1.5 H+ and Ni2+ differentially enhance a slow inactivation process that involves at least two inactivated states and that resting inactivation is probably a close variant of slow inactivation.
    The Journal of Physiology 08/2010; 588(Pt 16):3011-30. DOI:10.1113/jphysiol.2010.191544 · 5.04 Impact Factor
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