Channel KCNE1 Research Articles

Effects of IKs channel inhibitors in insulin-secreting INS-1 cells

Potassium channels regulate insulin secretion. The closure of K(ATP) channels leads to membrane depolarisation, which triggers Ca(2+) influx and stimulates insulin secretion. The subsequent activation of K(+) channels terminates secretion. We examined whether KCNQ1 channels are expressed in pancreatic beta-cells and analysed their functional role. Using RT/PCR cellular mRNA of KCNQ1 but not of KCNE1 channels was detected in INS-1 cells. Effects of two sulfonamide analogues, 293B and HMR1556, inhibitors of KCNQ1 channels, were examined on voltage-activated outwardly rectifying K(+) currents using the patch-clamp method. It was found that 293B inhibited 60% of whole-cell outward currents induced by voltage pulses from -70 to +50 mV with a concentration for half-maximal inhibition (IC(50)) of 37 microM. The other sulfonamide analogue HMR1556 inhibited 48% of the outward current with an IC(50) of 7 microM. The chromanol 293B had no effect on tolbutamide-sensitive K(ATP) channels. Action potentials induced by current injections were broadened and after-repolarisation was attenuated by 293B. Insulin secretion in the presence but not in the absence of tolbutamide was significantly increased by 293B. These results suggest that 293B- and HMR1556-sensitive channels, probably in concert with other voltage-activated K(+) channels, influence action potential duration and frequency and thus insulin secretion.

Impaired KCNQ1–KCNE1 and Phosphatidylinositol-4,5-Bisphosphate Interaction Underlies the Long QT Syndrome

Nearly a hundred different KCNQ1 mutations have been reported as leading to the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2) regulates the KCNQ1-KCNE1 complex. In the present study, we show that PIP2 affinity is reduced in three KCNQ1 mutant channels (R243H, R539W, and R555C) associated with the long QT syndrome. In giant excised patches, direct application of PIP2 on the cytoplasmic face of the three mutant channels counterbalances the loss of function. Reintroduction of a positive charge by application of methanethiosulfonate ethylammonium on the cytoplasmic face of R555C mutant channels also restores channel activity. The channel affinity for a soluble analog of PIP2 is decreased in the three mutant channels. By using a model that describes the KCNQ1-KCNE1 channel behavior and by fitting the relationship between the kinetics of deactivation and the current amplitude obtained in whole-cell experiments, we estimated the PIP2 binding and dissociation rates on wild-type and mutant channels. The dissociation rate of the three mutants was higher than for the wild-type channel, suggesting a decreased affinity for PIP2. PIP2 binding was magnesium-dependent, and the PIP2-dependent equilibrium constant in the absence of magnesium was higher with the wild-type than with the mutant channels. Altogether, our data suggest that a reduced PIP2 affinity of KCNQ1 mutants can lead to the long QT syndrome.