Peter Proks

Abstract:

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Abstract:

1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells.

Abstract:

Insulin secretion is triggered by a rise in the intracellular Ca2+ concentration that results from the activation of voltage-gated Ca2+ channels in the beta-cell plasma membrane. Multiple types of beta-cell Ca2+ channel have been identified in both electrophysiological and molecular biological studies, but it appears that the L-type Ca2+ channel plays a dominant role in regulating Ca2+ influx. Activity of this channel is potentiated by protein kinases A and C and is inhibited by GTP-binding proteins, which may mediate the effects of potentiators and inhibitors of insulin secretion on Ca2+ influx, respectively. The mechanisms by which elevation of intracellular Ca2+ leads to the release of insulin granules is not fully understood but appears to involve activation of Ca2+/calmodulin-dependent protein kinase. Phosphorylation by either protein kinase A or C, probably at different substrates, potentiates insulin secretion by acting at some late stage in the secretory process. There is also evidence that small GTP-binding proteins are involved in regulating exocytosis in beta cells. The identification and characterisation of the proteins involved in exocytosis in beta cells and clarification of the mechanism(s) of action of Ca2+ is clearly an important goal for the future.