Dual effects of intracellular magnesium on muscarinic potassium channel current in single guinea‐pig atrial cells.

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Abstract
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.