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Abstract:

1. A combination of patch-clamp, amperometric and fluorimetric methods were used to investigate the Ca2+ dependence and kinetics of secretion from pancreatic beta-cells elicited by voltage-gated Ca2+ entry. 2. Whether measured by the change in cell capacitance or by amperometric detection of 5-hydroxytryptamine (5-HT) release, the voltage dependence of the amount of secretion mirrored that of both the peak Ca2+ current and Ca2+ entry. 3. The magnitude of secretion elicited by a single pulse could be entirely accounted for by a readily releasable pool of approximately 200 vesicles. Neither depression nor potentiation of release was observed with 0.1 Hz pulse trains. 4. Transient amperometric currents were detected, which occurred independently of each other and were attributed to the fusion of single vesicles. 5. The time course of the macroscopic amperometric current could be accurately reconstructed by convolution of the all-events latency distribution and the unitary amperometric current. 6. In response to membrane depolarisation, secretion was initiated with a variable latency: approximately 95 % of the first secretory events occurred at least 50 ms after the start of the voltage pulse (and Ca2+ influx). Secretion fell rapidly on membrane repolarisation, even though the average intracellular calcium concentration ([Ca2+]i) was still elevated. 7. The [Ca2+] in the locality of the release site was estimated from the all-events latency distribution. [Ca2+] rose during a voltage pulse and secretion was elicited at > 0.4 microM and peaked at approximately 2-10 microM.

Abstract:

Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (K(ATP)) channels in the absence of nucleotides. K(ATP) channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mM), in the absence and presence of magnesium nucleotides, on different types of cloned K(ATP) channels expressed in Xenopus oocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by approximately 50% but rapidly activated Kir6.2/SUR2A ( approximately 4-fold) and Kir6. 2/SUR2B ( approximately 2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6. 2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates K(ATP) channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1-6 of SUR.

Abstract:

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction with a high-affinity site on SUR1 and a low-affinity site on Kir6.2. MgADP and diazoxide interact with SUR1 to promote channel activity. 2. We examined the effect of N-terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2/SUR1. 3. A 14 amino acid N-terminal deletion (DeltaN14) did not affect the gating, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, Kir6.2DeltaC26, expressed in the absence of SUR1. Thus, the N-terminal deletion does not alter the intrinsic properties of Kir6.2. 4. When Kir6.2DeltaN14 was coexpressed with SUR1, the resulting KATP channels had a higher open probability (Po = 0.7) and a lower ATP sensitivity (Ki = 196 microM) than wild-type (Kir6.2/SUR1) channels (Po = 0.32, Ki = 28 microM). High-affinity tolbutamide block was also abolished. 5. Truncation of five or nine amino acids from the N-terminus of Kir6.2 also enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high-affinity tolbutamide block, although to a lesser extent than for the DeltaN14 deletion. Site-directed mutagenesis suggests that hydrophobic residues in Kir6. 2 may be involved in this effect. 6. The reduced ATP sensitivity of Kir6.2DeltaN14 may be explained by the increased Po. However, when the Po was decreased (by ATP), tolbutamide was unable to block Kir6. 2DeltaN14/SUR1-K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166S/SUR1-K719A,K1385M currents (which in the absence of ATP have a Po of > 0.8 and are not blocked by tolbutamide). Thus the N-terminus of Kir6.2 may be involved in coupling sulphonylurea binding to SUR1 to closure of the Kir6.2 pore.

Abstract:

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[gamma-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[gamma-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 microM ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.