Regulation of ROMK by Extracellular Cations

The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (Kir1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10mM increased whole-cell outward conductance by a factor of 3.4±0.4 in 15min and by a factor of 5.7±0.9 in 30min (n=22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5±0.5 over a period of 40min (n=15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4±0.8 pS to 11.1±0.8 pS (10mM K, n=8) or 11.7±1.2 pS (Cs, n=8). There was no effect of either 10mM K or cesium on the high open probability (Po=0.97±0.01; n=12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50mM K. In cell-attached patches, exposure to 50mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (Kir2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.