Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes

Abstract

The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea-pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea-pig cardiac ventricle. A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea-pig genomic library. The cell-specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi-cell RT-PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. After transfection in human embryonic kidney cells (HEK293) the mean single-channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. Cell-attached measurements in isolated guinea-pig cardiomyocytes (n = 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half-maximum block at -100 mV were 3.24 M for gpKir2.1, 0.51 M for gpKir2.2, 10.26 M for gpKir2.3 and 235 M for gpKir2.4. Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell-attached recordings. The concentration and voltage dependence of Ba2+ block of the large-conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. Our results suggest that the large-conductance inward rectifier channels found in guinea-pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate-conductance (23.8 pS) and low-conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.

Figure 1. Genomic structure and cellular localization of guinea-pig Kir2 genes

A, schematic drawing of the gene structure; the hatched areas represent the coding regions. The length of the DNA in base pairs (bp) and the position of the introns (arrows) are indicated. B, Kir2 gene transcripts in cardiomyocytes and coronary endothelial cells. RT-PCR of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 in pure fractions of isolated cardiac muscle cells (upper panel) and capillary endothelial cells (lower panel) selected with the ‘cell picker’. Troponin T and endothelin-1 were used as specific markers of cardiac muscle cells and endothelial cells, respectively (Preisig-Müller et al. 1999b).

Figure 2. Cellular localization of Kir2.4 by immunofluorescence

Kir2.4 immunoreactivity was observed in the perikarya and in the neuropil of an epicardial parasympathetic ganglion (A) and in a dense fibre plexus around coronary arteries (B, arrows) as well as in axons between cardiomyocytes (B, arrowheads). The weak fluorescence exhibited in cardiomyocytes (B) represents a non-specific autofluorescence that is also seen in controls. C, bundles of non-myelinated Kir2.4-immunoreactive axons (arrows) in phrenic nerve. Scale bar, 20 μm.

Figure 6. Current-voltage relation of native and cloned inward rectifier channels

The mean single-channel current-voltage relations of gpKir2.1 (▪), gpKir2.2 (•) and gpKir2.3 (▴) expressed in HEK293 cells and the mean single-channel current-voltage relations of native inward rectifier channels found in cardiomyocytes are plotted. The native channels with a slope conductance in the range 8.5-13.5 pS (Δ), 21.5-25 pS (□) and 28-39 pS (○) were grouped together. The error bars indicate standard deviation.

Figure 3. Single-channel currents of gpKir2 expressed in HEK293 cells

A, typical cell-attached recording of a gpKir2.2 channel at transmembrane potentials between -120 and +80 mV. The K⁺ concentration in the bathing solution and in the pipette solution was 140 and 150 mm, respectively. No channel openings were seen at positive potentials. B, cell-attached patch-clamp recordings of gpKir2.1, gpKir2.2 and gpKir2.3 channels. The applied membrane potential was -100 mV (inside - outside). The single-channel amplitudes were 2.3, 3.9 and 1.2 pA, respectively.

Figure 4. Single-channel recordings in cardiomyocytes

Continuous cell-attached single-channel recordings lasting up to 20 min were performed with 150 mm K⁺ in the pipette solution. The cells were superfused with normal physiological salt solution. The transmembrane potential of the patch (shown above each record) was calculated as the difference between the applied pipette potential and the mean resting potential recorded in the whole-cell configuration. The resting potential was -72 ± 0.19 mV; n = 31. A, typical recording with two identical channels of 33.5 pS in the same patch. The corresponding amplitude histogram shows three narrow peaks. B, recording from another patch containing two channels. The corresponding amplitude histogram shows four peaks, indicating that the two channels had a different conductance (32 and 22 pS). C, recording from a patch containing two channels with conductance 32.5 and 10.5 pS.

Figure 5. Conductance histograms of single-channel recordings

The single-channel conductance was calculated by linear regression from cell-attached recordings obtained at transmembrane potentials of -80, -100 and -120 mV. None of the channels showed outward currents at positive transmembrane potentials. A, conductance histogram of the Kir2 channels expressed in HEK293 cells. B, conductance histogram of single-channel recordings in isolated cardiomyocytes with a pipette solution containing 150 mm K⁺. C, conductance histogram of single-channel recordings in isolated cardiomyocytes with a pipette solution containing 60 mm K⁺. This histogram includes all measurements without Ba²⁺ and with 0.2 or 1 μm Ba²⁺ in the pipette.

Figure 7. Ba²⁺ block of Kir2 channels expressed in Xenopus oocytes

A, typical record of the dependence of Ba²⁺ block of Kir2.1 on voltage and time. The extracellular K⁺ concentration was 60 mm. Voltage steps to +40, -20, -40, -60, -80 and -100 mV were applied from a holding potential of 0 mV. B, Ba²⁺ concentration-effect curves for gpKir2.1 (▪), gpKir2.2 (▴), gpKir2.3 (•) and gpKir2.4 (▪) were determined at -100 mV. The data were fitted with the function I_Ba/I_con= 1/(1 +[Ba²⁺]/K_d). I_con was defined as the difference between the inward rectifier current measured under control conditions and the current measured in the presence of a maximal concentration of Ba²⁺ (5 mm for gpKir2.1 and 1 mm for the other gpKir2 channels, which induced complete block of inward rectifier channels). Analogously, I_Ba was defined as the difference between the current measured in the presence of a given concentration of Ba²⁺ and the current measured in the presence of a maximal concentration of Ba²⁺.

Figure 8. Comparison of Ba²⁺ sensitivity in native and cloned inward rectifier channels

The voltage dependence of the K_d values for Ba²⁺ block of cloned Kir2 channels and of native cardiac inward rectifier channels is plotted on a semi-logarithmic scale. Filled symbols: the K_d for Ba²⁺ block of gpKir2.1 (▪), gpKir2.2 (▴), and gpKir2.3 (•) expressed in Xenopus oocytes, measured in the presence of 60 mm external K⁺. Open circles and dotted line: the K_d for Ba²⁺ block of the large-conductance inward rectifier channels in cardiomyocytes (calculated from the data shown in Fig. 9), also measured in the presence of 60 mm external K⁺. The lines are linear fits of logK_dversus membrane potential.

Figure 9. Block of single inward-rectifier channels by extracellular Ba²⁺

A, typical cell-attached single-channel recordings on isolated cardiomyocytes at -100 mV. The pipette solutions contained 60 mm K⁺ and 0, 0.2, 1 or 5 μm Ba²⁺ as indicated on the left. B, dependence of the open probability at -120, -100 and -80 mV on the Ba²⁺ concentration in the pipette. All channels with a conductance in the range 23.5-27.5 pS were included. The open probability was normalized to the mean open probability recorded at the same potential under control conditions (without Ba²⁺ in the pipette solution). The data were fitted with the function P_o(Ba)/P_o(control)= 1/(1 +[Ba²⁺]/K_d). The number of patches recorded under each condition is given in parentheses. In the absence of Ba²⁺ the open-state probability was 0.919 ± 0.004 at -80 mV (n = 8), 0.863 ± 0.02 at -100 mV (n = 11) and 0.848 ± 0.019 at -120 mV (n = 9).