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

Abstract

The effects of sulfhydryl reagents on M-type voltage-dependent potassium currents ( I K(M)) were examined in NG108-15 cells transformed to express m1 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 −30 mV, associated with an inhibition of I K(M). The glutathione-induced response was mimicked by cysteine. These effects were also reproduced by superfusion with micromolar concentrations of HgCl 2, AgNO 3, N-methylmaleimide and p-chloromercuribenzoic acid ( pCMB), agents which target protein thiols. Glutathione, HgCl 2, AgNO 3 and pCMB inhibited the peak conductance of I K(M) without shifting the half activating voltage ( V 1/2), which was comparable to the acetylcholine (ACh)-induced response. The voltage dependence of time constants for I K(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.