Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium

Abstract

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) >> K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) >> K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

Figure 1. Kir7.1 is highly expressed in the RPE

A, Northern blot analysis of total RNA (10 μg lane⁻¹) extracted from bovine RPE, retina and other tissues and hybridized with human Kir7.1 probe. B, the same RNA gel stained with ethidium bromide.

Figure 2. Characterization of macroscopic Kir7.1 currents

A, families of currents measured from a Kir7.1 cRNA-injected Xenopus oocyte bathed with standard (2 mm K⁺) solution in the absence (upper panel) and presence (middle panel) of 10 mm Ba²⁺. The horizontal line to the left of the records indicates the zero-current level. The voltage protocol used to evoke these currents is shown below. B, I-V relationships obtained from the same oocyte using the voltage-ramp method.

Figure 3. Dependence of Kir7.1 current and conductance on [K⁺]_o

A, current-voltage relationships obtained from a single Kir7.1 cRNA-injected oocyte bathed with various [K⁺]_o. Data were generated from currents evoked by voltage steps. B, relationship between zero current potential and [K⁺]_o. Each filled circle and vertical bar indicates the mean and s.e.m. for 12 oocytes. The continuous line shows the linear regression fit to the data. C, relationship between conductance and membrane voltage. Chord conductance was calculated from the data in A. D, relationship between conductance and driving force. The same data in C plotted as a function of V_m–E_K.

Figure 4. Effect of cation substitution on Kir7.1 conductance

A, macroscopic currents recorded from a representative Kir7.1 cRNA-injected oocyte bathed in 98 mm K⁺, Na⁺, Li⁺, Cs⁺, or Rb⁺. Currents were evoked by the voltage-step protocol indicated. B, current- voltage relationships obtained with various monovalent cations in the bath. Each point and vertical bar represents the mean ±s.e.m. for 9 oocytes. C, same data as in B but at a higher gain to show zero current potentials. Connecting lines have no theoretical significance.

Figure 5. Determination of the reversal potential of Kir7.1 currents from Ba²⁺-sensitive currents

Panels depict averaged I-V relationships obtained in the presence and absence of 10 mm Ba²⁺ with either 98 mm K⁺ (A), Rb⁺ (B), Na⁺ (C), Li⁺ (D) or Cs⁺ (E) as the sole monovalent cation (n = 5). Ba²⁺-sensitive currents are also depicted.

Figure 6. Non-stationary noise analysis of Kir7.1 Rb⁺ currents

A, family of currents recorded from a cell-attached patch on a Kir7.1 cRNA injected oocyte with 98 mm Rb⁺ and 1 mm Ba²⁺ in the pipette. The bath contained 98 mm K⁺ external solution to depolarize the membrane potential. B, I-V relationships obtained from the currents in A measured 2.5 ms (○) and 167 ms (○) after the onset of the voltage step. C, relationship between current variance (σ²) and mean current from the same patch as in A. See text for additional details.

Figure 7. Properties of the Kir current in the RPE

A, families of whole-cell currents recorded from an isolated bovine RPE bathed with standard (5 mm K⁺) solution in the absence (upper panel) and presence of 10 mm Ba²⁺ (middle panel). The voltage protocol used to evoke these currents is shown below. B, I-V relationships obtained from the same cell as in A using a voltage-ramp protocol.

Figure 8. Dependence of RPE Kir currents on [K⁺]_o

A, I-V relationships obtained from a single bovine RPE cell bathed with various [K⁺]_o. Data were generated from currents evoked by voltage steps. B, relationship between zero current potential and [K⁺]_o. Each filled circle and vertical bar indicates the mean ±s.e.m. for 8 cells. The continuous line shows the linear regression fit to the data. C, relationship between conductance and membrane voltage. Chord conductance was calculated from the data in A. D, relationship between conductance and driving force. The same data in C plotted as a function of V_m–E_K.

Figure 9. Effect of cation substitution on the RPE Kir conductance

A, macroscopic currents recorded from a representative bovine RPE cell bathed in 140 mm K⁺, Na⁺, Li⁺, Cs ⁺, or Rb⁺. Currents were evoked by the voltage-step protocol indicated. B, I-V relationships obtained with various monovalent cations in the bath. Each point and vertical bar represent mean and s.e.m. for 11 RPE cells. C, same data as in B but at a higher gain to show zero current potentials. Connecting lines have no theoretical significance.

Figure 10. Determination of the reversal potential of RPE Kir currents from Ba²⁺-sensitive currents

Panels depict averaged I-V relationships obtained in the presence and absence of 10 mm Ba²⁺ with either 140 mm K⁺ (A), Rb⁺ (B), Na⁺ (C), Li⁺ (D) or Cs⁺ (E) as the sole monovalent cation (n = 11). Ba²⁺-sensitive currents are also depicted.

Figure 11. Non-stationary noise analysis of RPE Kir Rb⁺ currents

A, family of currents recorded from a cell-attached patch on the apical membrane of a bovine RPE cell with 140 mm Rb⁺ and 1 mm Ba²⁺ in the pipette. The bath contained 140 mm K⁺ external solution to depolarize the membrane potential. B, I-V relationships obtained from the currents in A measured 2.5 ms (○) and 167 ms (•) after the onset of the voltage step. C, relationship between current variance and mean current from the same patch as in A. See text for additional details.