Kir7.1 disease mutant T153I within the inner pore affects K+ conduction

Abstract

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid side-chain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full-length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K⁺ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K⁺ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wild-type channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel's narrow inner pore in regulating conductance.

Keywords: Kir7.1; electrophysiology; innerpore structure; pediatric blindness; potassium channels.

Figure 1.

T153I mutation primarily alters Kir7.1 function. HEK293 cells were transfected with either GFP-Kir7.1 WT (A) or T153I (D). After fixation, the cells were stained with a C-terminal Kir7.1 antibody (C-12) (B and E). Merged images (C and F) signify the expression of Kir7.1 protein on the cell membrane. G: Kir7.1 WT (n = 8) current trace average from −150 to 50 mV voltage ramp for 5 mM K⁺ (black) and 135 mM Rb⁺ (dashed), the time-course plot of current amplitude at −150 mV from a single cell is in the insert. H: T153I (n = 9) current trace average from voltage ramp for 5 mM K⁺ (gray) and 135 mM Rb⁺ (dashed), the time-course plot of inward current amplitude at −150 mV from a single cell is in the insert. I: K⁺ current amplitude at −150 mV compares Kir7.1 wildtype and T153I (P < 0.0001). *Significance ≤ 0.05. Current from untransfected cells is included as the negative control in I and J. J: zero-current potential comparison between Kir7.1 wildtype and T153I (P < 0.0001) calculated from G and H. K: Rb⁺ current amplitude at −150 mV for Kir7.1 wildtype and T153I. HEK293, human embryonic kidney cells; Kir7.1, inward-rectifier potassium channel; K⁺, potassium; Rb⁺, rubidium; WT, wildtype.

Figure 2.

Mutant channel does not show extracellular K⁺ dependence. A: Kir7.1 K⁺ current from −150 to 50 mV voltage ramp in 5 mM (n = 9), 10 mM (n = 7), 50 mM (n = 8), and 100 mM (n = 8) external K⁺. Colors and symbols remain the same for A–E. B: T153I K⁺ current (gray) voltage ramp in 5 mM (n = 6), 10 mM (n = 2), 50 mM (n = 6), and 100 mM (n = 6) extracellular K⁺. C: untransfected cell K⁺ current voltage ramp in 5 mM (n = 5), 10 mM (n = 5), 50 mM (n = 4), and 100 mM (n = 4) extracellular K⁺. D: normalized chord conductance in 100 mM K⁺ [g/g_{(−150 mV)}] from A and B comparing wild-type Kir7.1 and T153I (n ≥ 3). Dashed line indicates untransfected cells (n ≥ 4) in C–E. E: plot of g/g_{(100 mM K⁺)} at −150 mV across external K⁺ concentration calculated from A and B (n ≥ 3). F: plot of zero-current potential relative to external K⁺ concentration for wild-type Kir7.1 (black) and T153I (gray) from A and B. Kir7.1, inward-rectifier potassium channel; K⁺, potassium.

Figure 3.

Effect of amino acid side chains on channel localization and K⁺ current. A: live-cell image of HEK293 cells expressing T153L, the inset is an intensity plot (A–E), and I-V plot showing average current recordings from 5 cells in HR (solid line) and Rb⁺ (dashed line) (n = 5). For all live-cell images, green is GFP, red is WGA-594 membrane stain, and blue is Hoescht nuclear stain. All intensity plots show fluorescence arbitrary unit (Fl. AU) vs. distance (Dist.) B: live-cell image of HEK293 cells expressing T153G, and average (n = 7) I-V plot in HR (solid line) and Rb⁺ (dashed line). C: live-cell image of HEK293 cells expressing T153A, and average (n = 7) I-V plot in HR (solid line) and Rb⁺ (dashed line). D: live-cell image of HEK293 cells expressing T153C, and average (n = 8) I-V plot in HR (solid line) and Rb⁺ (dashed line). E: live-cell image of HEK293 cells expressing T153S and average (n = 7) I-V plot in HR (solid line) and Rb⁺ (dashed line). F: average I-V plot as current traces in response to voltage ramp from −150 to 50 mV in 5 mM K⁺ of Kir7.1, T153I, T153L, T153G, T153A, T153C, and T153S. G: average I-V plot responses in 135 mM Rb⁺ for the same cells as in F. H: inward-current amplitude measured at −150 mV for alternate side-chain mutants compared with wildtype and T153I. *P value ≤ 0.05 compared with wildtype. I: zero-current potential calculated from F of alternate side-chain mutants compared with wildtype and T153I. J: Rb⁺ current amplitude at −150 mV from G. GFP, green fluorescent protein; HEK293, human embryonic kidney cells; HR, extracellular Ringer’s solution; Kir7.1, inward-rectifier potassium channel; K⁺, potassium; Rb⁺, rubidium; WGA594, wheat germ agglutinin/Alexa594 conjugate; WT, wildtype.

Figure 4.

Functional correlation of amino acid polarity and size with extracellular K⁺. A: average current trace for T153A channel (voltage ramp −150 to 50 mV) in 5 mM (n = 4), 10 mM (n = 4), 50 mM (n = 3), and 100 mM (n = 3) external K⁺. Symbols by concentration are the same for A–F. B: average current trace for T153C channel in 5 mM (n = 6), 10 mM (n = 6), 50 mM (n = 6), and 100 mM (n = 6) external K⁺. C: average current trace for T153S channel in 5 mM (n = 6), 10 mM (n = 6), 50 mM (n = 6), and 100 mM (n = 6) external K⁺. D: normalized chord conductance to 100 mM K⁺ [g/g_{(−150 mV)}] from A–C compared with WT and T153I across voltages (n ≥ 3). E: plot of g/g_{(100 mM K⁺)} at −150 mV across external K⁺ concentration from A and B compared with WT and T153I (n ≥ 3). F: plot of zero-current potential relative to external K⁺ concentration for polar and short side-chain T153 mutants compared with wildtype and T153I channel from A–C. K⁺, potassium; WT, wildtype.

Figure 5.

Molecular models of altered pore structure and size. A: top view of the Kir7.1 WT homology model with amino acid 153 in black. The black circle represents the hydrated K⁺ ion in A–E. The dashed arrow (shown in E) indicates the distance between the side chains used to measure the pore radius (PR). B: top view of the T153I mutant with amino acid 153 in red. C: top view of T153C with amino acid 153 in green. D: top view of T153S with amino acid 153 in orange. E: top view of T153A with amino acid 153 in purple. F: top view of T153G with amino acid 153 in blue. G: top view of T153L with amino acid 153 in yellow. H: membrane protein explorer (MPex) data from both Gibbs free energy (black) and hydropathy (gray) calculations for the wildtype Kir7.1, T153I, T153C, T153S, T153L, T153G, and T153A channels. Kir7.1, inward-rectifier potassium channel; WT, wildtype.

Figure 6.

Kir7.1 structure-function correlation. Functional channels form a narrow inner pore with two hydrogen bonds (dashed lines) between the amino acid side chains at position 153 and the backbone of amino acid 149, conducting both K⁺ and Rb⁺. Dysfunctional channels form a constricted inner pore with one hydrogen bond between the amino acid side chain at position 153 and the backbone of amino acid 149 that selectively restricts K⁺ conductance. Nonfunctional channels form a wide inner pore with one hydrogen bond between the amino acid side chain at position 153 and the backbone of amino acid 149. Channels with altered functions have limited K⁺ and Rb⁺ conductance. Kir7.1, inward-rectifier potassium channel; K⁺, potassium; Rb⁺, rubidium.