A.E. Busch

Hypotonic solution increases the slowly activating potassium current IsK expressed in Xenopus oocytes

A slowly activating potassium current was expressed in Xenopus oocytes by injection of RNA transcribed from a rat kidney cDNA clone. Hypotonic solutions (160 mOsmol/l; control was 220 mOsmol/l) increased the current by increasing the rate of activation and by decreasing the depolarization needed to activate the current. This effect of hypotonicity was not observed in calcium-free solution, but was unaffected by staurosporine or the calmodulin antagonist W7. Cytochalasin D reduced the current and prevented the increase by hypotonic solution. The results suggest that the increase in this potassium current by hypotonic solution might result from calcium entry and changes in the actin network.

Current inactivation involves a histidine residue in the pore of the rat lymphocyte potassium channel RGK5

RGK5 is a rat genomic DNA clone that encodes the n-type potassium channel found in T-lymphocytes and other cells. Current through this channel declines (inactivates) over a period of hundreds of milliseconds during a maintained depolarizing pulse, whether in lymphocytes or when expressed in Xenopus oocytes. Here we demonstrate that an amino acid residue near the outer pore of the channel, histidine401, is involved in the inactivation process. Replacement of this residue by tyrosine, the amino acid found in the equivalent position of the homologous but non-inactivating channel RBK1, reduced inactivation of RGK5 over a 5 s depolarizing pulse from 84.3 +/- 1.9% to 18.3 +/- 1.1%. Conversely, replacement of this tyrosine in RBK1 (Tyr379) by histidine increased its inactivation from 21.6 +/- 1.1% to 42.3 +/- 1.5%. These results suggest a mechanism of channel inactivation distinct from that previously described for the A-type potassium channel.

Persistent activation of min K channels by chemical cross-linking

Expression of the structurally and functionally distinct min K channel in Xenopus oocytes results in voltage-dependent potassium currents that activate with a characteristic slow time course. Application of a membrane-impermeable chemical cross-linking agent to oocytes expressing min K decreased the time-dependent current, increased its rate of activation, and induced persistently activated inward and outward potassium currents. These effects required membrane depolarization, demonstrating use dependence. Persistently activated channels retained potassium selectivity and sensitivity to block by clofilium and barium. These results suggest that a major conformational change occurs during min K channel gating, which can be stabilized by chemical cross-linking, and are consistent with a model in which min K channels activate by voltage-dependent subunit aggregation.