The purpose of this study was to test the hypothesis, based on indirect morphological, biochemical and physiological evidence, that glial cell AQP4 and Kir4.1 interact functionally. We found, by multiple criteria, no significant differences in Kir4.1 K+
channel function in freshly isolated glial cells from wild-type vs. AQP4 null mice, including resting cell membrane potential, whole-cell current analysis and single-channel analysis. Kir4.1 K+
channel function also did not differ in astroglial cell cultures from wild-type vs. AQP4 null mice. Further, we found no significant differences in AQP4 water permeability in primary glial cell cultures after barium inhibition of Kir4.1 function or RNAi knock-down of Kir4.1 expression. These results mandate the identification of alternative mechanisms to explain altered seizure and cortical spreading depression dynamics in AQP4 null mice, as well as the slowed K+
reuptake from the extracellular space following neuroexcitation (Binder et al., 2004a
; Padmawar et al., 2005
). The conclusions here are in agreement with our recent study showing unimpaired Kir4.1 K+
currents in retinal Müller cells from AQP4 knock-out mice (Ruiz-Ederra et al., 2007
). However, in that study it was not possible to study effects of Kir4.1 inhibition/knock-down on AQP4 water permeability. Further, demonstration of unimpaired Kir4.1 function in retinal Müller cells does not have compelling physiological relevance because the visual phenotype in AQP4 null mice was very mild (Li et al., 2002
), contrasting with the substantially more robust brain neuroexcitation phenotypes (Binder et al., 2004a
). Possible differences in composition and/or assembly of the proposed dystrophin complex in glial vs. retinal Müller cells have been proposed to account for the different phenotypes.
Functional measurements of Kir4.1 were done on freshly isolated astroglial cells, well before possible phenotype changes associated with cell culture might occur. The astroglial cells were isolated by enzymatic digestion from brain slices as reported before (Kimelberg et al., 2000
; Zhou and Kimelberg, 2001
). The isolated astroglial cells retained numerous processes as they display in intact brain. Several different types of K+
currents have been characterized in this model, including Kir4.1 channels (Kimelberg et al., 2000
). Viable astroglial cells were identified by SR101 staining, as reported for their labeling in in vivo
imaging and in vitro
brain slice preparations (Jourdain et al., 2007
; Nimmerjahn et al., 2004
We obtained a bimodal distribution of resting membrane potentials from the isolated astroglial cells, as found previously in hippocampus slices (D'Ambrosio et al., 1998
). There are discrete classes of astroglial cells in hippocampus that differ in morphology and electrophysiological properties (D'Ambrosio et al., 1998
). Kir4.1 channels are nearly fully open at resting potential, and as reported before, are expressed in about one-half of the astroglial cells (Higashi et al., 2001
). The bimodal distribution of resting membrane potentials thus accounts for the differences in Kir4.1 expression. This interpretation is supported by our data showing that inwardly rectified K+
currents were never seen in the depolarized cell population.
Patch-clamp analysis indicated characteristic Kir4.1 K+
channel currents in the freshly isolated glial cells, including weak inward rectification, as found for Kir4.1 currents recorded from Müller cells (Ishii et al., 1997
; Kofuji et al., 2000
; Ruiz-Ederra et al., 2007
), Kir4.1-transfected cells (Tada et al., 1998
) and cultured astroglia (our result). Also, from single channel analysis, the Kir4.1 channel in freshly isolated glial cells showed a single population with 20–25 pS unitary conductance at negative potentials, and 0.8–1 open probability, in agreement with recordings from Müller cells and Kir4.1-transfected cells (Ishii et al., 1997
; Ruiz-Ederra et al., 2007
). However, neither the function nor the expression of Kir4.1 differed in wild-type vs. AQP4 null mice.
Measurements of AQP4 water permeability were done in primary glial cell cultures from neonatal brain cortex, as we have established in prior studies on effects of AQP4 deletion on glial cell water permeability (Solenov et al., 2004
). Water permeability was measured by a calcein quenching method, which is based on the sensitivity of cytoplasmic calcein to cell volume. Glial cell cultures rather than isolated glial cells were used for these studies to allow for Kir4.1 RNAi knock-down and to give adequate signal-to-noise for accurate measurement of osmotic water permeability, which was not possible in freshly isolated, coverglass-immobilized glial cells. We found substantially more rapid osmotic equilibration in glial cell cultures from wild-type than from AQP4 null mouse brain; however, neither Kir4.1 inhibition by barium nor knock-down by RNAi affected AQP4 water permeability, indicating that the expression and function of AQP4 is independent of Kir4.1.
We conclude from these functional studies that altered Kir4.1 function in AQP4 deficiency does account for the mouse phenotype findings of altered seizure and cortical spreading depression dynamics in AQP4 deficiency, or of slowed K+
reuptake from brain extracellular space following neuroexcitation (Binder et al., 2006
; Padmawar et al., 2005
). Perhaps altered extracellular space volume or dynamics, which is supported by photobleaching measurements of macromolecular diffusion (Binder et al., 2004b
; Papadopoulos et al., 2005a
), is in part responsible for the mouse phenotype findings. Alternatively, other glial cell or neuronal ion transporters, whose expression or function are altered in AQP4 deficiency, may account for the phenotype findings.