Implication of Kir4.1 channel in excess potassium clearance: an in vivo study on anesthetized glial-conditional Kir4.1 knock-out mice

Abstract

The K(ir)4.1 channel is crucial for the maintenance of the resting membrane potential of glial cells, and it is believed to play a main role in the homeostasis of extracellular potassium. To understand its importance in these two phenomena, we have measured in vivo the variations of extracellular potassium concentration ([K(+)](o)) (with potassium-sensitive microelectrodes) and membrane potential of glial cells (with sharp electrodes) during stimulations in wild-type (WT) mice and glial-conditional knock-out (cKO) K(ir)4.1 mice. The conditional knockout was driven by the human glial fibrillary acidic protein promoter, gfa2. Experiments were performed in the hippocampus of anesthetized mice (postnatal days 17-24). Low level stimulation (<20 stimuli, 10 Hz) induced a moderated increase of [K(+)](o) (<2 mm increase) in both WT and cKO mice. However, cKO mice exhibited slower recovery of [K(+)](o) levels. With long-lasting stimulation (300 stimuli, 10 Hz), [K(+)](o) in WT and cKO mice displayed characteristic ceiling level (>2 mm increase) and recovery undershoot, with a more pronounced and prolonged undershoot in cKO mice. In addition, cKO glial cells were more depolarized, and, in contrast to those from WT mice, their membrane potential did not follow the stimulation-induced [K(+)](o) changes, reflecting the loss of their high potassium permeability. Our in vivo results support the role of K(ir)4.1 in setting the membrane potential of glial cells and its contribution to the glial potassium permeability. In addition, our data confirm the necessity of the K(ir)4.1 channel for an efficient uptake of K(+) by glial cells.

Figure 1.

K_ir4.1 cKO glial cells are more depolarized than WT glia. A, Example of an in vivo intraglial and DC field recordings in a WT mouse under anesthesia. The star marks the moment of electrode withdrawal from the cell. B, Box plot (left) and distribution (right) of K_ir4.1 cKO (n = 14 cells; gray histogram) and WT R_IN (n = 24 cells; white histogram). No significant difference was found (Mann–Whitney U test, p = 0.28). The central box covers the middle 50% of each sample; the sides of the box are the lower and the upper quartiles, and the vertical line drawn through the box is the median. The whiskers extend out to the lower and upper values of the samples. Outliers are represented as individual squares beyond the whiskers. The mean is presented with a cross. C, Box plot (left) and distribution (right) of K_ir4.1 cKO (n = 14 cells; gray histogram) and WT (n = 24 cells; white histogram) membrane potential. cKO glial cells are significantly more depolarized than WT glial cells (Mann–Whitney U test, p < 0.001). D, Absence of correlation between membrane potential and R_IN in both groups. Dots represent individual values, lines are linear fits of the former.

Figure 2.

K_ir4.1 cKO mice display impairment of extracellular potassium clearance. A, Hippocampal fast-green injection in one of the animals allowing the localization of the K⁺ recording electrodes. B, Representative recordings of [K⁺]_o responses induced by trains of stimulation in WT and K_ir4.1 cKO mice. The stimulations are indicated below the trace with rectangles. The inset depicts an average response to a train of 10 stimuli from one animal (black trace) and the exponential curve fitting of the former (red trace). C, The grand average trace of the exponential fittings after 5, 10, and 20 stimulations for WT (n = 12; black traces) and cKO (n = 10; red traces) groups. Circles indicate the respective time constants. D, Grand average of power spectra of local field potentials from the two groups of mice (WT, black trace; cKO, red trace; n = 5 in each group). The two power spectra are statistically distinct (p < 0.05) for the whole calculated range, with an additional difference within the 6–13 Hz frequency band (between dotted vertical lines) case in which the p values were <0.01 (Mann–Whitney U test).

Figure 3.

In vivo [K⁺]_o undershoot after long-lasting stimulations. Representative responses of the [K⁺]_o variations induced by long trains of stimulations (100, 200, 300, 600 stimuli at 10 Hz) in a WT mouse. Note the appearance of an undershoot (long K⁺ decrease below the basal level once stimulations stopped) with the increase of the duration of the stimulation. A clear undershoot starts to be visible with 300 stimulations. Top trace shows a representative EEG recording before and after long-lasting stimulations. In the central box, average of the first 10 (1) and last 10 (2) stimulations in the stimulation train. In the EEG recording, the epoch with stimuli has been skipped. In the [K⁺]_o traces, stimulation artifacts were artificially removed to illustrate the evolution of the [K⁺]_o dynamic.

Figure 4.

Long-lasting stimulation induces a larger [K⁺]_o undershoot in K_ir4.1 cKO mice. A, Comparison between representative [K⁺]_o response to 300 stimuli in WT and K_ir4.1 cKO mice. Note the larger undershoot induced in the recording from the cKO mouse (stimulation artifacts were removed). B, Description of the parameters considered for the comparison of the [K⁺]_o responses in WT and cKO mice (B1). The time constant was evaluated with an exponential curve fit between the end of the stimulation and the maximum amplitude of the undershoot. Maximum amplitudes of the [K⁺]_o during stimulations and during undershoot were evaluated with regard to the basal [K⁺]_o level. The duration of the undershoot (“time of return”) corresponds to the time needed for [K⁺]_o to recover to the basal level after the end of the stimulation. The total surface area of the undershoot was also estimated (gray area). No difference was found between WT and K_ir4.1 cKO recordings for [K⁺]_o clearance (as reflected by the time constant) and amplitude of [K⁺]_o responses (B2,B3). However, the [K⁺]_o undershoot is statistically more pronounced in the cKO. Area and amplitude of [K⁺]_o undershoot are significantly different between the cKO and WT (B4,B5). The time for the [K⁺]_o return to basal level is also longer in the cKO (B6, cKO mice: n = 5, WT mice: n = 5; Mann–Whitney U test, *p < 0.05, **p < 0.02).

Figure 5.

K_ir4.1 cKO glial cells display a decrease in potassium permeability. A, Representative traces from a WT glial cell showing parallel evolution of the cell's membrane potential and [K⁺]_o variations induced by a train of 300 stimuli. Bottom left panel shows a detail of the period during stimulation with the stimulation artifact removed, clearly demonstrating that WT glial cells behave as “[K⁺]_o sensitive-electrodes.” Bottom right panel shows cross-correlation ± SD between membrane potential of WT glial cells and [K⁺]_o dynamic (n = 6 recordings). The positive peak of the cross-correlation suggests in-phase relationship between the two waves, whereas its high amplitude (93.9%; n = 6 recordings) indicates the high degree of global resemblance of the two waves. B, Representative traces depicting decorrelation between the K_ir4.1 cKO glial cell membrane potential and [K⁺]_o induced by a train of 300 stimuli, suggesting a decrease in the cell's K⁺ permeability. C, The magnitude, but not the trend, of the cKO membrane potential response is dependent on the number of stimulations.