1. We have used excised inside-out patches to study the effects of anions bathing the cytoplasmic surface of the membrane on ATP-dependent K+ channels of rat flexor digitorum brevis muscle. Channels were closed by ATP applied to the cytoplasmic face of the patch with a concentration for half-closure (K1) of 14 ,UM, were highly selective for K+ and had unitary conductances of 62 pS in symmetrical 155 mm K+ and 27 pS in 5 mM[K+]o.2. In 139 mm Cl-internal solution channel activity declined rapidly after excision of the patch. Inclusion of 40 mm potassium gluconate (substituted for KCl) in the solution both restored channel activity and greatly slowed its subsequent run-down. 3. The action of gluconate was concentration dependent. The effect did not involve a change in ATP binding, since the K1 for ATP was not significantly changed by gluconate, and was specific for the cytoplasmic face of the patch. 4. The anions pyruvate, lactate and acetate were all able to restore channel activity after run-down, though less well than gluconate, while sulphate and methylsulphate were without effect. 5. Analysis of single channel kinetics showed that gluconate did not affect mean open lifetime, but led to a decrease in the number and duration of long closings. 6. Anions are most likely to act by stabilizing the structure of the channel protein.

1. We have studied the block by intracellular Mg21 (0-08-4 mM) of ATP-dependent potassium channels (KATP channels) from rat skeletal muscle using inside-out excised sarcolemmal patches. The block is voltage dependent, is relieved by extracellular potassium and has rapid kinetics, allowing the use of amplitude distribution analysis to estimate on and off rates. 2. To gain insight into the pore properties necessary to produce such a block, we have used an energy barrier model based on Eyring rate theory. (Findlay, 1987;Ashcroft & Kakei, 1989;Vivaudou, Arnoult & Villaz, 1991), but, secondly, Mg2+ produces a rapid voltagedependent block of the channel which causes mild inward rectification of the unitary current-voltage relation (i-V) at physiological intracellular Mg2+ concentrations (Findlay, 1987;Horie, Irisawa & Noma, 1987;Woll, Lonnendonker & Neumcke, 1989). Voltage-dependent channel block by cations is usually assumed to result from the blocking ion having to enter the channel, and so experience some of the transmembrane voltage field, in order to reach its binding site (Woodhull, 1973). On this basis Mg2+ appears to pass about 0-2-0-4 of the electrical distance into the channel to exert its fast block of KATP channels (Horie et al. 1987;Ciani & Ribalet, 1988). Mg2+ block of KATP channels can be relieved by increasing [K+]O (Horie et al. 1987).In the present paper, we report a detailed study of the voltage-dependent block by Mg2+ of KATP channels from rat skeletal muscle. We find that both voltage and extracellular K+ affect the off rate for Mg2+, and we have used rate theory analysis to develop a model that describes K+ permeation and the kinetics of Mg2+ block in this channel. METHODS PreparationAdult Wistar rats were killed by stunning followed by cervical dislocation, and the flexor digitorum brevis muscle was dissected from the hindlimb. To dissociate single fibres, the muscle was incubated in 0 3 % collagenase (Type 1; Sigma) in Ringer solution (containing (mM): NaCl, 146-3; KCI, 4-75; CaCl2, 1; CaHPO4, 0 95; MgCl2, 0 5; Hepes, 9.5, adjusted to pH 7-4 with NaOH) for 30 min at 4°C and then for 90 min at 37 'C. Fibres were subsequently isolated in collagenase-free Ringer solution by triturating the muscle with a fire-polished Pasteur pipette. Sarcolemmal vesicles were formed by placing the isolated fibres in a 155 mm K+ solution, and inside-out patches were excised from these into flowing intracellular solution containing 40 mM gluconate

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