Inward rectifier K+ channels mediate the K+ conductance at resting potential in many types of cell. Since these K+ channels do not pass outward currents (inward rectification) when the cell membrane is depolarized beyond a trigger threshold, they play an important role in controlling excitability. Both a highly voltage-dependent block by intracellular Mg2+ and an endogenous gating process are presently assumed to underly inward rectification. It is shown that strong voltage dependence of rectification found under physiological conditions is predominantly due to the effect of intracellular spermine. Physiological concentrations of free spermine mediate strong rectification of IRK1 inward rectifier K+ channels even in the absence of free Mg2+ and in IRK1 mutant channels that have no endogenous rectification.
Potassium (K+) homeostasis is controlled by the secretion of K+ ions across the apical membrane of renal collecting duct cells through a low‐conductance inwardly rectifying K+ channel. The sensitivity of this channel to intracellular pH is particularly high and assumed to play a key role in K+ homeostasis. Recently, the apical K+ channel has been cloned (ROMK1,2,3 = Kir1.1a, Kir1.1b and Kir1.1c) and the pH dependence of ROMK1 was shown to resemble closely that of the native apical K+ channel. It is reported here that the steep pH dependence of ROMK channels is determined by a single amino acid residue located in the N‐terminus close to the first hydrophobic segment M1. Changing lysine (K) at position 80 to methionine (M) removed the sensitivity of ROMK1 channels to intracellular pH. In pH‐insensitive IRK1 channels, the reverse mutation (M84K) introduced dependence on intracellular pH similar to that of ROMK1 wild‐type. A detailed mutation analysis suggests that a shift in the apparent pKalpha of K80 underlies the pH regulation of ROMK1 channels in the physiological pH range.
P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X 2 receptor splice variant (P2X2_2) carrying a 207 bp deletion in the intracellular Cterminus and the analysis of the corresponding genomic structure of the P2X 2 gene. P2X 2 _2 is as highly expressed as the original P2X 2 sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X 2 or P2X2_ 2 receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X 2 isoforms.
Large subtype-specific differences in the sensitivity of cloned inward-rectifier K' channels of the IRKl, BIRlO and ROMKl subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIRlO channels compared to ROMKl channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIRlO, is homologous to the residue in IRK1 and ROMKl which has previously heen shown to change gating properties and Me sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K' channels.
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