Differential pH sensitivity of Kir4.1 and Kir4.2 potassium channels and their modulation by heteropolymerisation with Kir5.1

Abstract

1. The inwardly rectifying potassium channel Kir5.1 appears to form functional channels only by coexpression with either Kir4.1 or Kir4.2. Kir4.1-Kir5.1 heteromeric channels have been shown to exist in vivo in renal tubular epithelia. However, Kir5.1 is expressed in many other tissues where Kir4.1 is not found. Using Kir5.1-specific antibodies we have localised Kir5.1 expression in the pancreas, a tissue where Kir4.2 is also highly expressed. 2. Heteromeric Kir5.1-Kir4.1 channels are significantly more sensitive to intracellular acidification than Kir4.1 currents. We demonstrate that this increased sensitivity is primarily due to modulation of the intrinsic Kir4.1 pH sensitivity by Kir5.1. 3. Kir4.2 was found to be significantly more pH sensitive (pK(a) = 7.1) than Kir4.1 (pK(a) = 5.99) due to an additional pH-sensing mechanism involving the C-terminus. As a result, coexpression with Kir5.1 does not cause a major shift in the pH sensitivity of the heteromeric Kir4.2-Kir5.1 channel. 4. Cell-attached single channel analysis of Kir4.2 revealed a channel with a high open probability (P(o) > 0.9) and single channel conductance of approximately 25 pS, whilst coexpression with Kir5.1 produced novel bursting channels (P(o) < 0.3) and a principal conductance of approximately 54 pS with several subconductance states. 5. These results indicate that Kir5.1 may form heteromeric channels with Kir4.2 in tissues where Kir4.1 is not expressed (e.g. pancreas) and that these novel channels are likely to be regulated by changes in intracellular pH. In addition, the extreme pH sensitivity of Kir4.2 has implications for the role of this subunit as a homotetrameric channel.

Figure 1. Immunohistochemical localization of Kir 5.1 subunits in the rat pancreas

A, Kir 5.1 immunoreactivity was detected in the islets of Langerhans (arrows) and also on the acinar cells (*). B, control slices show total absence of brown staining. C, an intense immunoreactivity is detectable in pancreatic arteriolae (arrow) and acinar cells (*). Scale bars: 25 μm in A and B; 10 μm in C.

Figure 2. Modulation of Kir4.1-Kir5.1 channel activity by acidification and alkalinisation

A, inside-out patch recordings from oocytes expressing Kir4.1-Kir5.1 channels. Channel openings were evoked by hyperpolarising pulses to -100 mV, from a holding potential of 0 mV. The representative trace shows the effect on channel activity by the application of a cytoplasmic solution at pH 8, through a multibarrel fast-flow pipette. B, whole-cell representative current traces recorded from a Xenopus oocyte expressing Kir4.1(K67M)-Kir5.1(M73K) channels. Currents were evoked by hyperpolarisation to -100 mV for 4 s from a holding potential of -10 mV followed by a 2 s pulse to +40 mV. Current traces are shown before (control) and after intracellular acidification to the indicated value (pH_i 6.4). C, intracellular pH vs. current inhibition for Kir4.1-Kir5.1 (○), Kir4.1 (•), Kir4.1(K67M)-Kir5.1 (△), Kir4.1(K67M)-Kir5.1(M73K) (♦) and Kir4.1(K67M) (▴). Data points were obtained from currents recorded at -100 mV in the TEVC configuration in control conditions and during the perfusion of a membrane-permeant potassium acetate buffer that reduces the oocyte intracellular pH to the indicated value (mean ±s.e.m. of 6-8 oocytes). The continuous line shows the fit with the equation 1/[1 + ([H⁺]_i - K)ⁿ] from which the apparent pK_a values were calculated. D, relative current inhibition (as recorded in B) that was induced by intracellular acidification to pH 6.3 for wild-type and mutant channels.

Figure 5. pH sensitivity of Kir4.2 and Kir4.2-Kir5.1 channels recorded in excised patches

pH vs. relative inhibition as determined by exposure of excised, inside-out giant membrane patches to different pH solutions.

Figure 3. Kir4.2 and Kir4.2-Kir5.1 currents are inhibited by intracellular acidification

A, representative whole-cell current traces recorded in the TEVC configuration from Xenopus oocytes expressing the indicated channels. The recordings were performed as detailed in Fig. 2B. C and D, I-V curves that were evoked by 1 s voltage ramps from -100 to +100 mV and were recorded using inside-out giant patches excised from oocytes expressing Kir4.2 (C) and Kir4.2-Kir5.1 (D) channels. The currents shown exhibit little rectification as they were were recorded in the absence of Mg²⁺ and polyamines and during the cytoplasmic application of solutions at the indicated pHs through a multibarrel fast-flow system. Strong rectification was observed in the cell-attached configuration before patch excision into Mg²⁺- and polyamine-free solution.

Figure 4

A, pH sensitivity of Kir4.2 and Kir4.2-Kir5.1 channels to intracellular pH. Intracellular pH vs. current inhibition determined in TEVC configuration for Kir4.2 (•), Kir4.2-Kir5.1 (○) and Kir4.2(K66M) (▴). B, increased sensitivity of Kir4.2 correlates with the C-terminal region. Intracellular pH vs. current inhibition determined as in A for Kir4.1 (○) and Kir4.2 (•) compared to chimeras 31B (▪) and 31C (△) which exchange the intracellular C-terminus (see inset). The difference in sensitivity between the two subunits correlates with the C-terminus.

Figure 6. Single-channel properties of homomeric Kir4.2

A, representative cell-attached patch recording at -100 mV from an oocyte expressing Kir4.2 channels. The bottom record is shown on an expanded time scale. B, current-voltage relationship of Kir4.2. A slope conductance of 25.2 pS was calculated from the fit with a linear regression line. The extrapolated zero-current potential was approximately -14 mV. Each data point represents the average channel current calculated from amplitude histograms (mean ±s.d. of 6 patches pulled from different oocytes).

Figure 7. Single-channel properties of heteromeric Kir4.2-Kir5.1 channels

A, representative cell-attached patch recording at -100 mV from an oocyte expressing tandemly linked Kir4.2-Kir5.1 channels. The bottom record is shown on an expanded time scale. B, current-voltage relationship of Kir4.2-Kir5.1 channel. A slope conductance of 54.2 pS was calculated from the fit with a linear regression line. The extrapolated zero-current potential was approximately -30 mV. Each data point represent the average channel current calculated from amplitude histograms (mean ±s.d. of 6 patches pulled from different oocytes).