Dystrophin Dp71 is critical for the clustered localization of potassium channels in retinal glial cells

Abstract

The Müller cell is the principal glial cell of the vertebrate retina. The primary conductance in Müller cells is the inwardly rectifying potassium channel Kir4.1 (BIR10 and KAB-2), which is highly concentrated at the endfeet at the vitreal border and to processes enveloping blood vessels. Such asymmetric and clustered distribution of Kir4.1 channels in Müller cells is thought to be critical for the buffering of extracellular potassium concentration in retina. Herein we investigated whether the distribution and functional properties of Kir4.1 channels are dependent on expression of the Dp71, a dystrophin isoform expressed in Müller cells. Kir4.1 distribution was determined in mouse retinal sections and whole mounts using anti-Kir4.1 antibodies and confocal microscopy. In Müller cells from wild-type mice, Kir4.1 is highly clustered in their endfeet and perivascular processes. In contrast, in Müller cells from the mdx(3Cv) mouse, which lacks the expression of Dp71, the Kir4.1 immunoreactivity is evenly distributed throughout the cell membrane. Surface expression of Kir4.1 is not affected in mdx(3Cv) Müller cells as current density of barium-sensitive inward currents in mdx(3Cv) Müller cells are not different from wild type. Focal extracellular potassium increases in isolated Müller cells shows that Kir channels in the mdx(3Cv) cells, as opposed to wild type, are less prominently concentrated in their endfeet. In summary, our data indicate that Dp71 is critical for the clustering but not membrane expression of Kir4.1 in mouse Müller cells. These results point to a new role for dystrophin in glial cells.

Fig. 1.

Kir4.1 localization in wild-type (wt) and mdx^3Cv retinal sections.A, Kir4.1 is concentrated at the inner limiting membrane (arrow) and to processes around blood vessels in wild-type retina (arrowheads). B, In the mdx^3Cv mouse, Kir4.1 appeared to be evenly distributed throughout the retina, and there appeared to be a reduction in staining at the inner limiting membrane (arrow) and no apparent enrichment of Kir4.1 around blood vessels (arrowheads). The Müller-specific marker glutamine synthetase (GS; C, D) and merged images (merged; E,F) suggest the localization of Kir4.1 to Müller cells. POS, Photoreceptor outer segments;ONL, outer nuclear layer; INL, inner nuclear layer. G, H, Retinal sections from wild type and mdx^3Cv were stained for GLAST. The structural integrity of Müller cells and their fine processes in the mutant mouse appeared to be intact and indistinguishable from that of the wild type. Scale bar, 25 μm.

Fig. 2.

Kir4.1 localization in wild-type (wt) and mdx^3Cv whole-mount retinal tissue. In both mice, optical sections were taken from the OPL (A, B), the IPL (C,D), the GCL (E, F), and the inner limiting membrane (ILM; D,H). In the wild-type mouse (A,C, E, G), Kir4.1 was prominently detected in processes surrounding blood vessels (green) in the OPL, IPL, and GCL, respectively, whereas glutamine synthetase staining (red) revealed the location of Müller cells. Colocalization is indicated byyellow areas. Kir4.1 staining is prominently displayed at the inner limiting membrane of the wild type (G). Arrowheads (B,D, F) delineate blood vessels present in the OPL, IPL, and GCL, respectively, of the mdx^3Cv mouse that were not noticeably stained for Kir4.1. Uneven staining intensity in G andH resulted from the retina not being completely flat. Scale bar, 25 μm.

Fig. 3.

A, A Western blot was performed using brain lysates from wild-type (wt) and mdx^3Cv mice and was exposed to an antibody specific for the hydrophobic C terminus of dystrophin. The lack of immunodetection in the mdx^3Cv lane confirms a lack of Dp71 in the mutant mouse. B, A Western blot showing expression levels of Kir4.1 in wild-type versus mdx^3Cv brain and retina. A band at ∼200 kDa represents Kir4.1 in its tetrameric form. Brain tissue from the Kir4.1 knock-out (KO) mouse was used as a negative control (far right lane). There was no distinguishable difference in Kir4.1 expression between wild-type and mdx^3Cv mouse.

Fig. 4.

Müller cell whole-cell electrophysiology in wild-type (wt) and mdx^3Cv mouse.A, Representative traces from Müller cells recorded in extracellular solutions containing no BaCl₂(left), 100 μm BaCl₂(middle), and 1 mm BaCl₂(right). B, Current–voltage relationships of the recordings in A. C, Resting membrane potential (V_m) was measured in wild-type (black bars) and mdx^3Cv (white bars) dissociated Müller cells in normal extracellular solution (2.5 K) and in the same solution with the addition of 100 μm BaCl₂ (100 Ba) or 1 mm BaCl₂ (1 Ba).D, Current density was calculated by first subtracting the evoked current at −120 mV with blockade by 1 mm BaCl₂ from the evoked current at −120 mV with 2.5 K extracellular solution in each cell. This value was then divided by membrane capacitance measured under blockade by 1 mm BaCl₂. (Membrane capacitance was recorded under barium blockade based on the increased ability to obtain a more accurate exponential fit under such high membrane resistance.) E, Currents at −120 mV were recorded in whole-cell clamp mode with a 400 msec pulse of −120 mV from a holding potential of −80 mV. The data point was taken 392 msec from the start of the voltage step. x-Axis labeling as in C. There were no significant current differences in wild type versus mdx^3Cv inC–E.

Fig. 5.

Regional K⁺ conductance is affected in mdx^3Cv Müller cells.A, Representative traces from whole-cell clamped Müller cells to which focal ejections of a 50 mmK⁺ solution were applied. The Müller cell diagram on the left is oriented such that the apical end is at the top and the endfoot is at thebottom, and the left and right columns show K⁺-evoked depolarization traces from wild-type and mdx^3Cv mice, respectively. Note the differences between cells with respect to current amplitudes in the endfoot and proximal stalk (regions 2, 3, and 4).B, Peak currents in each cell region were normalized to their respective peak endfoot current, and these values for all cells in each condition were averaged and plotted. The wild-type cells (filled circles; n = 9) maintained smaller normalized conductances in regions 2 (0.62 ± 0.20) and 3 (0.65 ± 0.31) in the proximal stalk, whereas the mdx^3Cv mouse (open circles;n = 8) demonstrated a larger normalized conductance in regions 2 (1.41 ± 0.36) and 3 (1.84 ± 0.74). *p < .05.