Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH

CO2 chemoreception may be related to modulation of inward rectifier K+ chan nels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. T o understand the biophysical and molecular mechanisms underlying the modula tion of these currents by CO2 and pH, heteromeric Kir4.1-Kir5.1 were studie d in inside-out patches. These Kir4.1-Kir5.1 currents showed a single chann el conductance of 59 pS with open-state probability (P-open) similar to 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was complete ly suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these curren ts was due to selective suppression of P-open without evident effects on si ngle channel conductance, leading to a decrease in the channel mean open ti me and an increase in the mean closed time. At pH 8.5, single-channel curre nts showed two sublevels of conductance at similar to 1/4 and 3/4 of the ma ximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents wee modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) tha t enhanced baseline P-open and reduced channel sensitivity to intracellular protons. In the presence of 10 mu M PIP2, the Kir4.1-Kir5.1 showed a pKa v alue of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely e liminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4 .1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K6 7M mutation remained sensitive to low pH(i). Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the tit ration curve of wild-type and mutant channels was totally abolished when Ar g178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric K ir channel that can be modulated by both acidic and alkaline pH, show the m odulation of pH sensitivity of Kir channels by PIP2, and provide informatio n of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.