Sulfhydryl modification inhibits K+ (M) current with kinetics close to acetylcholine in rodent NG108-15 cells

The effects of sulfhydryl reagents on M-type voltage-dependent potassium currents (IK(M)) were examined in NG108-15 cells transformed to express ml muscarinic acetylcholine receptors (mAChRs), a NGPM1-27 clone. Focal application of glutathione at millimolar concentrations dissolved in acidic solutions caused a transient inward current in NGPM1-27 cells at holding potentials of -30mV, associated with an inhibition of IK(M). The glutathione-induced response was mimicked by cysteine. These effects were also reproduced by superfusion with micromolar concentrations of HgCl2, AgNO3, N-methylmaleimide and p-chloromercuribenzoic acid (pCMB), agents which target protein thiols. Glutathione, HgCl2, AgNO3 and pCMB inhibited the peak conductance of IK(M) without shifting the half activating voltage (V1/2), which was comparable to the acetylcholine (ACh)-induced response. The voltage dependence of time constants for IK(M) deactivation in sulfhydryl reagent-, ACh- and non-treated cells resembled, but differed from that in Ba(2+)-treated cells. These results reveal that there is an accessible cysteine moiety, but not a disulfide bond, either on the M channel protein itself or on a protein directly involved in agonist-M channel coupling.