Abstract
Indirect evidence suggests that the Müller/glial cell water channel aquaporin-4 (AQP4) modulates K(+) channel function of the closely associated Kir4.1 protein. We used patch clamp to compare Kir4.1 K(+) channel function in freshly isolated Müller cells from retinas of wild-type (+/+) and AQP4 knock-out (-/-) mice. Immunocytochemistry showed a comparable Kir4.1 protein expression pattern in Müller cells from +/+ and -/- retinas, with greatest expression at their end feet. Osmotic water permeability was >4-fold reduced in -/- than in +/+ Müller cells. Resting membrane potential did not differ significantly in +/+ versus -/- Müller cells (-64 +/- 1 versus -64 +/- 1 mV, S.E., n = 24). Whole-cell K(+) currents recorded with a micropipette inserted into the cell soma were Ba(2+)-sensitive and showed no significant differences in magnitude in +/+ versus -/- Müller cells (1.3 +/- 0.1 versus 1.2 +/- 0.1 nA at -160 mV) or in inwardly rectifying current-voltage relationships. Spatially resolved K(+) currents generated by pulsed K(+) injections along Müller cell bodies were also comparable in +/+ versus -/- Müller cells. Single-channel cell-attached patch clamp showed comparable unitary conductance, current-voltage data, and open probability in +/+ versus -/- Müller cells. Thus, contrary to the generally accepted view, our results provide direct evidence against functionally significant AQP4 modulation of Müller cell Kir4.1 K(+) channel function.
Highlights
Calizes with the inwardly rectifying Kϩ channel Kir4.1 [4] and has been shown by immunoprecipitation to interact with AQP4 [5], likely as part of a macromolecular complex that includes ␣-syntrophin and dystrophin [6]
The distribution of Kir4.1 was not affected by AQP4 deletion, as seen qualitatively in Fig. 1A and by quantitative image analysis in Fig. 1B, where background-subtracted, area-integrated fluorescence intensity was determined along the axis of many isolated Muller cells and normalized to fluorescence in the soma
We found no influence of AQP4 deletion on Muller cell Kir4.1 function or distribution as measured by whole-cell current and single channel patch clamp recordings in freshly isolated Muller cells as well as by immunofluorescence in isolated cells and retinal sections
Summary
Calizes with the inwardly rectifying Kϩ channel Kir4.1 [4] and has been shown by immunoprecipitation to interact with AQP4 [5], likely as part of a macromolecular complex that includes ␣-syntrophin and dystrophin [6]. It is, generally believed without direct evidence that AQP4 modulates Kir4.1 Kϩ channel function. The purpose of this study was to test the hypothesis that AQP4 expression modulates Kir4.1 Kϩ channel function in retinal Muller cells. We found, based on multiple criteria, no influence of AQP4 knock-out on Muller cell Kir4.1 expression or function
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