Recently we have shown that expression in Xenopus oocytes of a member of the CLC family of chloride channels, ClC-0, elevates pHi (Cooper & Fong, 1998). Hypertonic shock activates a Na+/H+ exchanger endogenous to Xenopus oocytes (Humphreys et al. 1995). Exposing oocytes to hypertonic conditions also activates an anion conductance. We believe that the low native Cl– permeability is the rate-limiting step for KCl efflux across the oocyte membrane. Expressing ClC-0 removes this barrier; resulting in loss of KCl, cell shrinkage and Na+/H+ exchanger activation. To test this hypothesis, we manipulated the transmembrane K+ gradient and measured the resulting oocyte pHi.
Female Xenopus laevis were anaesthetised by immersion in 2 % tricaine, as directed by NIH guidelines. Ovarian lobes were surgically removed and stage V-VI oocytes were isolated by collagenase digestion (Cooper & Boron, 1998). Oocytes were injected with 1 ng of cRNA encoding ClC-0 or an equivalent volume of H2O (50 nl) on the day of isolation. Experiments were performed on the following day. The pHi and ClΔ were measured using ion-sensitive microelectrodes. The control oocyte bath solution (ND96) contained (mM): NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1 and Hepes 5. Unless otherwise specified, all solutions were adjusted to a pH of 7.50 and an osmolarity of 200 ± 10 mosmol. Values are quoted as means ± S.E.M. and statistical significance has been assumed at the 5 % limit.
ClC-0 expression was evident from the shift in the membrane potential from -52.0 ± 2.3 mV (n = 29) compared with -34.7 ± 1.0 mV (n = 25, P < 0.001) in control oocytes. Oocytes were incubated overnight in control ND96, or modified versions in which [K+] was set at 0.1 or 25 mM, and the resting pHi was measured in each group. For both control oocytes and oocytes expressing ClC-0, there was a linear relationship between log [K+] and pHi. However, the slope of this relationship increased from -0.05 in control oocytes to -0.1 in ClC-0-injected oocytes. These results support our hypothesis that the oocyte Cl– permeability is the limiting factor in movement of KCl across the cell membrane.
- Cooper, G.J. & Boron, W.F. (1998). Am. J. Physiol. 275, C1481-1486.
Cooper, G.J. & Fong, P. (1998). FASEB J. 12, A373.
Humphreys, B.D., Jiang, L., Chernova, M.N. & Alper, S.L. (1995). Am. J. Physiol. 268, C201-209.