Biophysical and Molecular Mechanisms Underlying the Modulation of Heteromeric Kir4.1–Kir5.1 Channels by Co2 and Ph

Abstract

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (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. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4.1–Kir5.1 were studied in inside-out patches. These Kir4.1–Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P open) ∼ 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P open without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at ∼1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1–Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P open and reduced channel sensitivity to intracellular protons. In the presence of 10 μM PIP2, the Kir4.1–Kir5.1 showed a pKa value 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 eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1–Kir5.1. In excised patches, interestingly, the Kir4.1–Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

Kir4.1/Kir5.1 channel forms the major K+channel in the basolateral membrane of mouse renal collecting duct principal cells

“…On the other hand, the coexpression of Kir4.1 and Kir5.1 units leads to the formation of a 43-to 60-pS K ϩ channel (34, 49) with an intermediate inward rectification (G in /G out ϳ4) and a P o of ϳ0.4 (49). In addition, channels formed by the Kir4.1/Kir5.1 assemblage are sensitive to pH i changes around the physiological range, with pK values of ϳ7.…”

Secretory modulation of basolateral membrane inwardly rectified K+ channel in guinea pig distal colonic crypts

“…Modulation of ROMK (Kir1.1; KCNJ1) via alkalinization increases P o by decreasing the number of long-duration closures without changing open durations (9,36). Heteromeric Kir4.1-Kir5.1 (KCNJ10-KCNJ16) also increases P o upon alkalinization through a loss of long-duration closures (59). Stimulation of IK1 (KCNN4) by the K ϩ channel opener 1-EBIO occurs through a decrease in long closed durations without any change in open durations (48).…”

Differential Effects on Current Kinetics by Point Mutations in the lurcher Motif of NR1/NR2A Receptors

“…Drawing analogy to pH-sensitive gating of potassium channels (Schulte and Fakler, 2000;Yang et al, 2000), we hypothesized that the molecular determinants for the proton site are localized near the extracellular end of the M3 transmembrane domain of NMDA receptors that contains the lurcher motif (SYTANLAAF). The lurcher motif is conserved in all glutamate receptors and is thought to play a critical role in gating (Kohda et al, 2000;Taverna et al, 2000;Jones et al, 2002).…”