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. 2008 Jan;37(1):1-10.
doi: 10.1016/j.mcn.2007.08.007. Epub 2007 Aug 15.

Aquaporin-4 independent Kir4.1 K+ channel function in brain glial cells

Hua Zhang  1 , A S Verkman
Affiliations

Aquaporin-4 independent Kir4.1 K+ channel function in brain glial cells

Hua Zhang et al. Mol Cell Neurosci. 2008 Jan.

Abstract

Functional interaction of glial water channel aquaporin-4 (AQP4) and inwardly rectifying K+ channel Kir4.1 has been suggested from their apparent colocalization and biochemical interaction, and from the slowed glial cell K+ uptake in AQP4-deficient brain. Here, we report multiple lines of evidence against functionally significant AQP4-Kir4.1 interactions. Whole-cell patch-clamp of freshly isolated glial cells from brains of wild-type and AQP4 null mice showed no significant differences in membrane potential, barium-sensitive Kir4.1 K+ current or current-voltage curves. Single-channel patch-clamp showed no differences in Kir4.1 unitary conductance, voltage-dependent open probability or current-voltage relationship. Also, Kir4.1 protein expression and distribution were similar in wild-type and AQP4 null mouse brain and in the freshly isolated glial cells. Functional inhibition of Kir4.1 by barium or RNAi knock-down in primary glial cell cultures from mouse brain did not significantly alter AQP4 water permeability, as assayed by calcein fluorescence quenching following osmotic challenge. These studies provide direct evidence against functionally significant AQP4-Kir4.1 interactions in mouse glial cells, indicating the need to identify new mechanism(s) to account for altered seizure dynamics and extracellular space K+ buffering in AQP4 deficiency.

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Figures

Figure 1
Figure 1. Kir4.1 and AQP4 expression in mouse brain and isolated astroglial cells
A, AQP4 immunoblot in wild-type (+/+) and AQP4 null (−/−) mouse hippocampus. β-actin immunoblot shown for same samples. B, (left) Kir4.1 and β-actin immunoblot in brain hippocampus and cortex. (right) Kir4.1 expression normalized to β-actin expression (SE, n=3, differences not significant). C, Immunofluorescence of AQP4 (red) and GFAP (blue). Bar, 10 μm. D, Immunofluorescence of AQP4 (red) and Kir4.1 (green) in isolated cells from mouse hippocampus (nuclei stained blue with DAPI). Bar, 10 μm.
Figure 2
Figure 2. Whole-cell currents in isolated astroglial cells in wild-type mice
A, Representative recordings in extracellular solutions containing 5 or 20 mM KCl, or 5 mM KCl + 100 μM Ba2+. Voltage steps were applied from a holding potential of − 80 mV to de- and hyper-polarizing potentials between − 180 and +60 mV (250 ms, 10 mV increments). Inset at lower right shows SR-101-stained astroglial cells and unlabeled pyramidal neuron (overlays of phase-contrast and fluorescence) (top) and fluorescence micrograph of a freshly isolated astrocyte during recording (bottom). B, Current-voltage (I–V) curves of whole-cell currents for a single astroglial cell at different [K+]o (5, 10 and 20 mM). Where indicated 100 μM Ba2+-was present in the extracellular solution. Data representative of 7 preparations. C, Whole-cell current elicited with a ramp protocol (left) with and without 100 μM Ba2+ and deduced I–V curve of Ba2+–sensitive current (right). Representative of 8 preparations.
Figure 3
Figure 3. Comparable whole-cell K+ currents in astroglial cells from wild-type and AQP4 null mice
A, Distribution of the resting membrane potentials (V) of SR101-positive cells isolated from wild-type (n=34, top) and AQP4 null (n=28, bottom) mice. Fit shown to bimodal Gaussian distributions. B, Mean whole-cell current-voltage curves in the hyperpolarized subpopulation of astroglial cells mice (S.E., n=17-21). Differences not significant. C, Mean voltage-ramp currents (S.E., n=8 per group). Differences not significant. Inset. Currents recorded by stepping potential from − 80 to − 160 mV for 30 ms, followed by a ramp to +40 mV over 300 ms.
Figure 4
Figure 4. Single-channel patch-clamp of Kir4.1 K+ channels in freshly isolated astroglial cells
A, Single-channel currents from cell-attached membrane patches of wide-type and AQP4 null astroglial cells with 145 mM K+ in the pipette. Holding potentials indicated to the left of each trace (c, channel closed level; o, channel open level). B, Unitary current-voltage data (S.E., n=4). Differences not significant. Dashed line shows linear regression of the inward current with inward single-channel conductances of 20 ± 3 pS (wild-type) and 21 ± 4 pS (AQP4 null). C, Open probability (Po) (S.E., n=6). Differences not significant. Where indicated, 100 μM Ba2+ was present in the pipette solution. Data are from one set of experiments representative of three. D, Increased Kir4.1 channel closed time in the presence of 100 μM Ba2+ (holding potential − 100 mV).
Figure 5
Figure 5. Characterization of astroglial cell cultures
A, (left) GFAP immunofluorescence of confluent wild-type and AQP4 null astroglial cell cultures (green). Bar, 10 μm. (right panels) AQP4 (red) and Kir4.1 (green) immunofluorescence. Nuclei counterstained blue using DAPI. Bar, 100 μm. B, AQP4 and Kir4.1 immunoblot, with β-actin used to normalize expression. Representative of 3 sets of culture. C, Single-channel current traces from cell-attached membrane patches at indicated holding potentials. Unitary current-voltage data shown, with dashed line giving unitary conductance 21.7 pS for inward currents. Data representative of 6 sets of preparations. D, Voltage steps applied from a holding potential of − 80 mV to de- and hyper-polarizing potentials between − 140 and +40 mV (250 ms, 10 mV increments). Ba2+-sensitive Kir4.1 currents computed from difference before and after addition of 100 μM Ba2+ (S.E., n=5). Differences not significant.
Figure 6
Figure 6. Kir4.1 inhibition or knock-down does not affect AQP4 water permeability
A, (left) Kir4.1 immunoblot of wild-type astroglial cell cultures at 3 days after treatment with Kir4.1 or control RNAi. β-actin used to normalize expression. (right) Reduced Kir4.1 expression after RNAi (S.E, n=3). B, Whole-cell currents in control and Kir4.1 RNAi-treated wild-type astroglial cells. (left) Currents shown in response to voltage steps from a holding potential of − 80 mV to de- and hyper-polarizing potentials between -160 and +60 mV (250 ms, 10 mV increments). (right) Reduced current density (pA/pF) in Kir4.1 RNAi-treated astroglial cells in response to voltage step from − 80 mV to − 160 mV (S.E., n=8). C, Osmotic water permeability. (left) Time course of calcein fluorescence in response to exchange between isosmolar and hypoosmolar (150 mOsm) solutions. Data shown for wild-type cell cultures under control conditions, and after Kir4.1 RNAi treatment or 100 μM Ba2+. Data for AQP4 null cells shown for comparison. (right) Summary of relative water permeability (S.E; n=8, *P < 0.001). Differences not significant among wild-type cultures.
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