Three methionine residues located within the regulator of conductance for K+ (RCK) domains confer oxidative sensitivity to large-conductance Ca2+-activated K+ channels.
Methionine-directed oxidation of the human Slo1 potassium channel (hSlo1) shifts the half-activation voltage by -30 mV and markedly slows channel deactivation at low concentrations of intracellular Ca2+ ([Ca2+]i). We demonstrate here that the contemporaneous mutation of M536, M712 and M739 to leucine renders the channel functionally insensitive to methionine oxidation caused by the oxidant chloramine-T (Ch-T) without altering other functional characteristics. Coexpression with the auxiliary beta1 subunit fails to restore the full oxidative sensitivity to this triple mutant channel. The Ch-T effect is mediated specifically by M536, M712 and M739 because even small changes in this residue combination interfere with the ability to remove the oxidant sensitivity following mutation. Replacement of M712 or M739, but not M536, with the hydrophilic residue glutamate largely mimics oxidation of the channel and essentially removes the Ch-T sensitivity, suggesting that M712 and M739 may be part of a hydrophobic pocket disrupted by oxidation of non-polar methionine to the more hydrophilic methionine sulfoxide. The increase in wild-type hSlo1 open probability caused by methionine oxidation disappears at high [Ca2+]i and biophysical modelling of the Ch-T effect on steady-state activation implicates a decrease in the allosteric coupling between Ca2+ binding and the pore. The dramatic increase in open probability at low [Ca2+]i especially within the physiological voltage range suggests that oxidation of M536, M712 or M739 may enhance the Slo1 BK activity during conditions of oxidative stress, such as those associated with ischaemia-reperfusion and neurodegenerative disease, or in response to metabolic cues.
Santarelli
Lindsey Ciali
LC
Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Wassef
Ramez
R
Heinemann
Stefan H
SH
Hoshi
Toshinori
T
D002460:Cell Line; D002700:Chloramines / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics*; D007930:Leucine / Q000235:genetics; D008564:Membrane Potentials; D008715:Methionine / Q000235:genetics; D008954:Models, Biological; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014162:Transfection
D002700:Chloramines; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D016877:Oxidants; D008715:Methionine; D007930:Leucine