The ion selectivity of a membrane conductance inactivated by extracellular calcium in Xenopus oocytes

The ion selectivity of a membrane ion conductance that is inactivated by extracellular calcium (Cao2+) in Xenopus oocytes has been studied using the voltage-clamp technique. The reversal potential of the Cao2+-sensitive current (Ic) was measured using voltage ramps (−80 to +40 mV) as a function of the external concentration (12–240 mM) of NaCl or KCl. The direction and amplitude of the shifts in reversal potentials are consistent with permeability ratios of 1:0.99:0.24 for K+:Na+:Cl−. Current-voltage (I–V) relations of Ic, determined during either voltage ramps of 0.5 s duration or at steady state, displayed pronounced rectification at both hyperpolarized and depolarized potentials. However, instantaneous I–V relations showed less rectification and could be fitted by the constant field equation assuming the above K+:Na+:Cl− permeability ratios. Ion substitution experiments indicated that relatively large organic monovalent cations and anions are permeant through Ic channels with the permeability ratios K+:NMDG+:TEA+:TPA+:TBA+:Gluc− = 1:0.45:0.35:0.2:0.2:0.2. External amiloride (200 μM), gentamicin (220 μM), flufenamic acid (40 μM), niflumic acid (100 μM), Gd3+ (0.3 μM) or Ca2+ (200 μM) caused reversible block of Ic without changing its reversal potential. Preinjection of oocytes with antisense oligonucleotide against connexin 38, the Xenopus hemi-gap-junctional protein, inhibited Ic by 80 % without affecting its ion selectivity, thus confirming and extending the recent suggestion of Ebihara that Ic represents current carried through hemi-gap-junctional channels. In vitro and in vivo maturation of oocytes resulted in a significant decrease in Ic conductance to 7 % and 2 % of control values, respectively. This developmental downregulation of Ic minimizes any toxic effect Ic activation would have when the mature egg is released into Cao2+-free pond water. The results of this study are discussed in relation to other Cao2+-inactivated conductances seen in a wide variety of cell types and which have previously been interpreted as arising either from Cao2+-masked channels or from changes in the ion selectivity of voltage-gated Ca2+ or K+ channels. For amphibian oocytes it has long been recognized that removal of extracellular calcium (Cao2+) from the bathing solution results in membrane depolarization (Tupper & Maloff, 1973; Dascal, 1987). The current underlying this depolarization has been referred to as Ic (Arellano, Woodward & Miledi, 1995). The events associated with this depolarization are toxic to immature oocytes since they lose viability and rupture within 12 h of incubation in zero-Ca2+ Ringer solution. Despite Ic being a large and consistently observable endogenous current, attempts to analyse its ion selectivity have given conflicting results. Originally Tupper & Maloff (1973), using tracer flux techniques, concluded that the Cao2+-sensitive depolarization in Ranapipiens arose through a large increase in permeability to Na+ with little contribution from K+ and Cl−. More recently, two groups using voltage-clamp techniques have analysed Ic in Xenopus oocytes. One group concluded that Ic was a cation-selective conductance which did not distinguish between Na+ and K+ (Arellano et al. 1995). The other group concluded that Ic was a Cl−-selective conductance with no apparent permeability for Na+ and K+ (Weber, Liebold, Reifarth, Uhr & Clauss, 1995a, b;Reifarth, Amasheh, Clauss & Weber, 1997). These apparently contradictory results may indicate that heterogeneities exist in the population of Ic channels in Xenopus oocytes such that Ic is mediated by subsets of cation- and anion-selective channels. In this case, under specific circumstances, one subset of channels might be masked so that the other subset dominates. Clearly the ion selectivity of Ic has important implications both for the identity of the underlying channel mechanism(s) and for comparison with other Cao2+-masked conductances seen in a variety of cell types, including epithelium (Van Driessche, Simaels, Aelvoet & Erlij, 1988), skeletal muscle (Almers, McCleskey & Palade, 1984), cardiac muscle (Guo, Ono & Noma, 1995; Mubagwa, Stangl & Flemeng, 1997), neurone (DeVries & Schwartz, 1992) and lymphocytes (Grissmer & Cahalan, 1989). Specifically, Arellano et al. (1995) initially considered that the Ic channel may be a hemi-gap-junctional channel (DeVries & Schwartz, 1992) or possibly the Gd3+-sensitive mechanically gated (MG) cation channel studied extensively in oocyte patch-clamp recordings (for review see Hamill & McBride, 1996). However, they ruled out both possibilities, the former because of the lack of anion permeability of Ic and the latter because of the Ca2+-independent activation and fast adaptation of MG channel activity in Xenopus oocytes (Hamill & McBride, 1992). Because of our long term interest in single MG channels and their as yet unresolved contribution to macroscopic oocyte currents, we were interested in resolving the underlying basis of the Ic conductance and, additionally, in testing the above subset channel hypothesis for Ic. Therefore, as a first step we have re-examined the ion selectivity of the macroscopic Ic. We followed two basic approaches to identify the ion species contributing to Ic, both involving measurement of reversal potential shifts. In one case, the shifts occur as a result of changes (i.e. increase or decrease) in the external NaCl (or KCl) concentration. In the other case, the shifts occur as a result of ion substitutions with large organic ions, expected to have reduced or zero permeation. In both approaches the reversal potential shifts can be compared with theoretical predictions for ideally selective cation- or anion-selective channels (see Appendix). As a second step in testing the subset channel hypothesis, we have made attempts to block selectively one of the channel subsets. If Ic is mediated by independent cation- and anion-selective channels, one would expect to see shifts in Ic reversal potential as one subset of channels is selectively blocked. We have tested a number of agents that are commonly used to block either cation channels (e.g. amiloride and gentamicin, which like Gd3+ block MG cation channels (Hamill & McBride, 1996)), or anion channels (e.g. flufenamic, niflumic and 9-anthracene carboxylic acids; Pappone & Lee, 1995). A preliminary account of this study has been presented to the Biophysical Society (Zhang, McBride & Hamill, 1997).