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 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.
Claydon
Thomas W
TW
Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC, Canada.
Vaid
Moni
M
Rezazadeh
Saman
S
Kwan
Daniel C H
DC
Kehl
Steven J
SJ
Fedida
David
D
D000818:Animals; D005456:Fluorescent Dyes; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials; D008845:Microinjections; D008954:Models, Biological; D009154:Mutation; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation; D012235:Rhodamines; D051637:Shaker Superfamily of Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D005456:Fluorescent Dyes; D012235:Rhodamines; D051637:Shaker Superfamily of Potassium Channels; C005358:tetramethylrhodamine; D011188:Potassium