Dual effects of intracellular magnesium on muscarinic potassium channel current in single guinea-pig atrial cells.
1. The effects of internal Mg2+ ions on the muscarinic acetylcholine (ACh) receptor-mediated K+ currents were investigated in single atrial cells of guinea-pigs, using the whole-cell and inside-out modes of the patch-clamp technique. 2. During cell dialysis in the whole-cell-clamp condition, the depletion of internal Mg2+ increased outward muscarinic K+ currents but decreased inward currents, thereby reducing the inwardly rectifying property of the channels. 3. When inside-out patches were prepared, channel availability was abolished and was reactivated by internal application of guanosine 5'-triphosphate (GTP) or its non-hydrolysable analogue, 5'-guanylyl imidodiphosphate (GppNHp), in the presence of Mg2+. GppNHp led to a recovery of the channels also in the nominal absence of Mg2+ (0[Mg2+]i). 4. The activation of single-channel currents by intracellular GTP and Mg2+ was dose-dependent. Both concentration-response curves were fitted by saturation kinetics with Hill coefficients of 1, and the half-maximum doses were 24 +/- 8 microM for GTP and 67 +/- 14 microM for Mg2+. The effects of Mg2+ on activation of K+ currents were additive with those of GTP, suggesting the presence of two independent binding sites for GTP and Mg2+. 5. The single-channel conductance became virtually ohmic when measured at nominally zero [Mg2+]i while GppNHp was used to recover the channel activity. Micromolar [Mg2+]i reduced the unitary amplitude of single open-channel currents in a dose- and voltage-dependent manner, showing half-blocking doses of 293 microM at +40 mV and 115 microM at +60 mV. 6. Voltage-dependent kinetics of Mg2+ block were described using equations based on Eyring rate theory (Woodbury, 1971; Hille, 1984), where the coefficient for voltage dependence (delta) was 0.63. 7. Intracellular Mg2+, at a physiological concentration, has a dual action on the muscarinic K+ channel: first Mg2+ activates the channel in the presence of GTP through GTP-binding proteins (G proteins), and secondly it blocks outward currents through the channel, thereby causing the inwardly rectifying property.
National Institute for Physiological Sciences, Okazaki, Japan.
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D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D005260:Female; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D006325:Heart Atria; D008274:Magnesium / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000502:physiology
D015221:Potassium Channels; D011976:Receptors, Muscarinic; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium; D000109:Acetylcholine