The effect of intracellular anions on ATP‐dependent potassium channels of rat skeletal muscle.

Abstract

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.

Anionic Phospholipids Activate ATP-sensitive Potassium Channels

“…These results suggest that although phosphorylation may be a part of the regulatory process, additional key elements are involved. In rat skeletal muscle cells, KATP was found to be activated by high concentrations (Ͼ50 mM) of gluconate, with the effect proportional to the length of the carbon chain (13). This finding suggested to us the hypothesis that native anionic FIG.…”

“…Mg 2ϩ was found to be necessary for ATP activation of KATP; therefore, many researchers speculated that phosphorylation/dephosphorylation was the key to maintenance of KATP channel activity (16). Mg-ATP, however, was often ineffective in activating KATP in rat skeletal muscles (13). In addition, intensive searches for protein kinases and the protein phosphorylation targets have not been successful (32).…”

“…Single ventricular myocytes were isolated from rabbit hearts by an enzymatic (0.1% collagenase, Williton) dissociation method (17,18). Single skeletal muscle cells were dissociated enzymatically (0.3% collagenase, Williton) from the flexor digitorum brevis muscles of the hind legs of Wistar rats (13). Isolated single skeletal muscle cells were suspended in Dulbecco's modified Eagle's medium until they were used.…”

“…KATP, however, also depends on another less well characterized regulatory process, because KATP gradually inactivates when the integrity of the cell is disrupted by, for example, the excised patch method of voltage clamp (11)(12)(13). This property is shared not only by most KATP channels from different tissues, but also by other channels of the inward rectifier potassium (Kir) superfamily (for reviews, see Refs.…”

Discrimination between subtypes of apamin‐sensitive Ca2+‐ activated K+ channels by gallamine and a novel bis‐quaternary quinolinium cyclophane, UCL 1530

“…We think this unlikely for several reasons. In the first place, the structurally related compound, dequalinium, has previously been shown not to block voltage-gated Ca2" channels in sympathetic neurones at concentrations up to 10 gM (Dunn, 1994 (Habermann & Fischer, 1979;Hugues et al, 1982;Habermann, 1984) and more recent work has shown that intracellular anions also affect the activity of a number of K+ channels, as well as their sensitivity to blocking agents (Zhang et al, 1994;Heath & Terrar, 1994;McKillen et al, 1994 (Wadsworth et al, 1994). This evidence is clearly consistent with the existence of SKCa channel subtypes.…”