CO2-dependent opening of an inwardly rectifying K+ channel

Abstract

CO(2) chemosensing is a vital function for the maintenance of life that helps to control acid-base balance. Most studies have reported that CO(2) is measured via its proxy, pH. Here we report an inwardly rectifying channel, in outside-out excised patches from HeLa cells that was sensitive to modest changes in PCO(2) under conditions of constant extracellular pH. As PCO(2) increased, the open probability of the channel increased. The single-channel currents had a conductance of 6.7 pS and a reversal potential of -70 mV, which lay between the K(+) and Cl(-) equilibrium potentials. This reversal potential was shifted by +61 mV following a tenfold increase in extracellular [K(+)] but was insensitive to variations of extracellular [Cl(-)]. The single-channel conductance increased with extracellular [K(+)]. We propose that this channel is a member of the Kir family. In addition to this K(+) channel, we found that many of the excised patches also contained a conductance carried via a Cl(-)-selective channel. This CO(2)-sensitive Kir channel may hyperpolarize excitable cells and provides a potential mechanism for CO(2)-dependent inhibition during hypercapnia.

Fig. 1

Increasing PCO₂ increases open probability of a small conductance channel. a Continuous record of the effects of different CO₂ concentrations on channel gating in outside-out patches from HeLa cells. Note that the effect of changing PCO₂ from the control level of 35 mmHg to the levels marked on the black bars on channel gating was rapid. Isolated patch was held at +10 mV. b Expanded traces from a demonstrating the gating of the channels in the patch. Dotted lines represent different levels of channel openings, and multiple openings are only seen at the higher levels of PCO₂. c All-points histograms obtained from the data in b and fitted with sums of Gaussian distributions to estimate P _o. d Plot of P _o vs PCO₂ (n = 5 for each point; bars are SEMs). Continuous line is drawn to the Hill equation, P _o  = 1/(1 + (45/PCO₂)²)

Fig. 2

Channel open time distributions recorded at different levels of PCO₂. Analysis of raw data from Fig. 1. The solid line is the combined fit of two exponential distributions, each shown separately as grey dashed lines. At each level of PCO₂, the fitting of two exponential distributions gave a statistically significantly better fit than a single exponential (P < 0.02, F test). The effects of 55 and 70 mmHg on channel open times are probably underestimated as these were taken from stretches of data early in the application of the elevated PCO₂ to minimize the occurrence of multiple-channel openings

Fig. 3

Current–voltage characteristics and permeability of the CO₂-sensitive channel. a Single-channel gating in outside-out excised patches during a series of 10 mV steps from +20 mV to –70 mV (left to right) in control 3 and 30 mM K⁺ aCSF at a PCO₂ of 70 mmHg. b Current–voltage plots of the single-channel currents in 3 mM K⁺ (n = 4) and 30 mM K⁺ (n = 4) aCSF. The reversal potential changed from –70 mV to –9 mV and the slope conductance of the channel was increased by elevating extracellular K⁺. c Plot of the open probability (P _o) against membrane potential for three excised patches, measured in 30 mM K⁺ aCSF. P _o exhibited no voltage dependence. Bars are SEMs

Fig. 4

Colocalization of Cl^– and K⁺ channels in excised patches. a Current–voltage relationship of single-channel currents recorded an outside-out patch in the presence of 15 mM Cl^– in the medium at a PCO₂ of 70 mmHg. The holding potential was changed from +40 to –70 mV in 10-mV steps (left to right). The inset shows the summary graph comparing the I–V relations for control (n = 7, black squares) and lowered Cl^– (open circles, n = 3). Bars are SEMs. b A continuous current record from the patch before and during the application of low Cl^– aCSF. Portions of the record in control aCSF (i) and low Cl^– aCSF (ii) are shown below. Note that the reduction of extracellular Cl^– ions reduces both the holding current and basal noise. Outside-out patch held at +10 mV