CCCP evokes an increase in [K⁺]_i in cultured astrocytes. A, under standard PBFI loading conditions, superimposed records of the changes in [K⁺]_i (represented by changes in cytosolic PBFI-derived BI₃₄₀/BI₃₈₀ ratio values) were evoked by four consecutive 2-min applications of 10 μm CCCP. For each response, BI₃₄₀/BI₃₈₀ ratio values were baseline adjusted to the resting BI₃₄₀/BI₃₈₀ value observed immediately prior to the appropriate application of CCCP. The rises in [K⁺]_i evoked by CCCP remained unaltered during repeated 2-min applications of CCCP (n = 5 preparations, p > 0.05). B, an image of astrocytes (excitation wavelength 340 nm) after preferentially loading of PBFI into mitochondria and following 50 μg/ml of saponin treatment to release cytosolic PBFI. Under these conditions signals largely emanate from mitochondria. C, in saponin-treated astrocytes under external Na⁺- and K⁺-free recording conditions, mitochondrial-derived PBFI BI₃₄₀/BI₃₈₀ ratio values declined rapidly upon uncoupling with a 2-min application of 10 μm CCCP indicating mitochondria release K⁺, which can be detected by cytosolic PBFI as indicated in A. D, under standard PBFI loading conditions prolonged application of 10 μm CCCP caused an immediate increase in [K⁺]_i followed by a gradual decrease in [K⁺]_i to a new steady state level. All records in A and D were obtained at [K⁺]_o = 3 mm.

K⁺ uptake mechanisms in cultured astrocytes. A, a representative trace of the effect of pretreatment with a K⁺ channel-blocking mixture on the rise in [K⁺]_i evoked by 12.5 mm K⁺_o in the continuous presence of CCCP. B, increases in [K⁺]_i evoked by 5-min applications of 12.5 mm [K⁺]_o during mitochondrial uncoupling with CCCP were significantly reduced in the presence of either 50 μm Ba²⁺ or a mixture containing 5 mm 4-AP, 3 mm Ba²⁺, and 1 mm TEA (*, p < 0.05 in each case), whereas no reduction was observed in the presence of 1 mm ouabain or 1 μm glyburide (p > 0.05). C, a representative trace displaying the BI₃₄₀/BI₃₈₀ transients (representing [K⁺]_i) evoked by 2-min applications of CCCP prior to and after a 5-min pretreatment with 12.5 mm K⁺_o. The K⁺ channel blocking mixture significantly attenuated the expected increase in the magnitude of the [K⁺]_i transient evoked by CCCP after exposure to 12.5 mm K⁺_o (compare with Fig. 3A). D, the effects of ouabain, voltage-activated K⁺ channel inhibitors, omeprazole, glyburide, 5-HD, and diazoxide in experiments of the type shown in C. All test compounds, except diazoxide and omeprazole, significantly reduced the 12.5 mm [K⁺]_o-induced increase in the CCCP response (*, p < 0.05). E, the effect of CCCP application on intracellular ATP levels (nanomole/mg of protein normalized to control). A 2-min application of CCCP followed by a 12-min wash and a 10-min application of CCCP significantly reduced ATP levels (*, p < 0.05). The respective n values are indicated in parentheses. Bars indicate the mean ± S.E.

K⁺ uptake in mitochondria from cultured astrocytes. A, mitochondria were preferentially loaded with PBFI and treated with saponin to remove cytosolic dye yielding signals largely derived from mitochondria. B, mitochondria were initially perfused with a K⁺-free solution followed by application of a solution containing 25 mm K⁺. In Cx43^+/+ cultures application of 25 mm K⁺ caused an increase in K⁺ in the mitochondria, an effect that was reduced by the Cx43 blocker CBX (20 μm). C, a graph quantifying the changes in K⁺ uptake rate, defined by the change in ratio value over the first 100 s during the initial rise in PBFI ratio upon application of 25 mm K⁺. K⁺ uptake was significantly reduced with treatment of the Cx43 blockers CBX (20 μm) and AGA (10 μm), and was not reduced with the inactive analogue GZA (20 μm). K⁺ uptake was also reduced in mitochondria obtained from Cx43^−/− mice. Application of CBX or AGA was ineffective in further reducing K⁺ uptake in Cx43^−/− mice. *, p < 0.05, n values in parentheses represent the number of individual coverslips tested. Bars indicate the mean ± S.E.

Mitochondrial uptake of K⁺ is reduced in Cx43^−/− astrocytes. A, a representative record of a prolonged CCCP application experiment depicting a rise in [K⁺]_i in response to 12.5 mm K⁺_o application to Cx43^−/− astrocytes. B, increases in [K⁺]_i evoked by 5-min applications of 12.5 mm K⁺_o during mitochondrial uncoupling with CCCP were not significantly different (p > 0.05) in Cx43^+/+, Cx43^+/−, or Cx43^−/− astrocytes. C, a representative trace displaying the [K⁺]_i transients evoked by 2-min applications of CCCP in the absence of and after 5 min pretreatment with 12.5 mm K⁺_o in Cx43^−/− astrocytes. D, using the same protocol as in C, Cx43^−/− astrocytes were found to have a reduced CCCP-induced [K⁺]_i response after high [K⁺]_o exposure in comparison to Cx43^+/+ astrocytes (*, p < 0.05), whereas Cx43^+/− astrocytes displayed an intermediate response. CBX or AGA were without effect in Cx43^−/− astrocytes (p > 0.05). The respective n values are indicated in parentheses. Bars indicate the mean ± S.E.

In situ calibration of PBFI. A, a calibration experiment in which PBFI-loaded cultured astrocytes on a single coverslip were exposed to 10 μm gramicidin-containing HEPES-buffered solutions at the [K⁺]_o values (in mm) shown above. B, a plot of [K⁺] versus PBFI-derived BI₃₄₀/BI₃₈₀ ratio values normalized to unity at [K⁺] = 100 mm from calibration experiments of the type shown in A. In comparison to the standard calibration curve (closed circle, n = 6), curves did not differ at [Na⁺]_o = 65 mm (open circle, n = 5), nor when pH was altered to pH 6.5 (closed square, n = 5), pH 8 (open square, n = 5), or in the presence of CCCP (open triangle, n = 5). In all cases, p > 0.05 (two-way ANOVA). Bars indicate the mean ± S.E.

Effects of changes in [K⁺]_o on CCCP-induced [K⁺]_i rises. A, an initial 2-min application of CCCP (at [K⁺]_o = 3 mm) evoked a [K⁺]_i transient. Following the recovery of [K⁺]_i to near resting levels, [K⁺]_o was increased to 12.5 mm for 5 min, immediately after which CCCP was again applied at [K⁺]_o = 3 mm. The rise in [K⁺]_i evoked by the second application of CCCP (indicated by b) was larger than that evoked by the first application (indicated by a) of the protonophore. B, the same experiment as illustrated in A, except [K⁺]_o was increased to 12.5 mm prior to the first application of CCCP. C, summary of the results obtained in experiments of the type shown in A and B conducted under HEPES- (open bars) and HCO₃⁻/CO₂⁻- (solid bars) buffered conditions. Also shown are results obtained under HEPES-buffered recording conditions at 37 °C (hatched bars) and with FCCP (crosshatched bars). In all cases, changes in [K⁺]_o (0–25 mm) were applied for 5 min immediately prior to the application of CCCP at [K⁺]_o = 3 mm. Results are presented as percent changes in the peak amplitudes of CCCP-evoked PBFI-derived BI₃₄₀/BI₃₈₀ ratio value transients (representing [K⁺]_i) after altered [K⁺]_o, compared with control responses evoked by CCCP in the absence of a preceding change in [K⁺]_o. In all cases, there was a significant change (*, p < 0.05) in the amplitude of [K⁺]_i transients observed after pretreatment with altered [K⁺]_o compared with those evoked under control conditions. Responses due to 12.5 mm [K⁺]_o were significantly different under HEPES- versus bicarbonate-buffered conditions (p < 0.05). Furthermore, responses to 12.5 mm [K⁺]_o under HEPES-based recording conditions were similar to the response in the FCCP and 37 °C groups (p > 0.05). D, following an initial 2-min application of CCCP at [K⁺]_o = 3 mm, astrocytes were exposed to 12.5 mm [K⁺]_o for 5 min, after which [K⁺]_o was reduced to 3 mm for 0, 30, or 60 s prior to the second application of CCCP (also at 3 mm K⁺_o). The increase in the CCCP-induced [K⁺]_i transient observed following pretreatment with 12.5 mm K⁺_o was not significantly altered (p > 0.05) at 30 s but was effectively abolished (*, p < 0.05) at 60 s after the end of the pretreatment (n = 5 in each case). E, exposure to 12.5 mm [K⁺]_o during mitochondrial uncoupling with prolonged CCCP treatment caused an increase in resting [K⁺]_i. F, summary of the results obtained in experiments of the type shown in E. Results are presented as the average amplitudes of the changes in PBFI-derived BI₃₄₀/BI₃₈₀ ratio values observed over the final 1-min exposure to altered [K⁺]_o after subtraction of the [K⁺]_i value observed immediately prior to altering [K⁺]_o. *, p < 0.05 in all cases. The respective n values are indicated in parentheses. Bars indicated the mean ± S.E.

Cx43 association with astrocyte mitochondria. An immunoblot of Cx43 expression in whole cell (WC) and mitochondrial (Mito) fractions of Cx43^+/+ (+/+) astrocyte cultures. Also indicated are the plasma membrane marker N-cadherin, the mitochondrial marker OxPhos, and GAPDH.

Mitochondrial uptake of K⁺ is reduced in the presence of gap junction blockers. A, pretreatment with the gap junction blocker CBX (20 μm) did not attenuate the rise in [K⁺]_i in response to the application of 12.5 mm K⁺_o in the continued presence of CCCP. B, changes in [K⁺]_i evoked by 5-min applications of 12.5 mm K⁺_o during mitochondrial uncoupling with CCCP were not reduced in the presence of 20 μm CBX, 10 μm AGA, or 20 μm GZA (p > 0.05 in all cases, compared with control). C, a representative record displaying a reduction in the [K⁺]_i transient evoked by a 2-min application of CCCP after 5 min pretreatment with 12.5 mm K⁺_o in the presence of AGA. D, using the same protocol illustrated in C, the effects CBX and GZA were also examined. All test compounds, except GZA, significantly attenuated (*, p < 0.05) the expected augmentation of CCCP-induced rises in [K⁺]_i following exposure to 12.5 mm K⁺_o. The respective n values are indicated in parentheses. Bars indicate the mean ± S.E.

Mitochondrial membrane potential during elevated [K⁺]_o in Cx43^+/+ and Cx43^−/− astrocytes. A, changes in ΔF/F values in rhodamine-123-loaded Cx43^+/+ and Cx43^−/− astrocytes upon exposure to 12.5 mm K⁺_o. B, bar graph summarizing the response to 12.5 mm [K⁺]_o in Cx43^+/+ and Cx43^−/− astrocytes. The amplitudes of responses to elevated [K⁺]_o were reduced in Cx43^−/− astrocytes (*, p < 0.05). The respective n values are indicated in parentheses. Bars indicate the mean ± S.E.