Effects of internal Na+ on the Ca channel outward current in mouse neoplastic B lymphocytes

The whole-cell configuration of the patch-clamp technique was used to study the outward Na+ current through Ca channels in hybridoma cell lines (202B and 206), constructed by fusion of S194 myeloma cells with murine splenic B lymphocytes. The concentration of Na+ in the electrode solution, [Na+]p, was changed by isosmotic replacement of Na+ with N-methyl-D-glucamine+ ions. When 2.5 mM calcium was present in the bath, neither the current nor the reversal potential was significantly altered by changes in the level of external Na+ [( Na+]o. By contrast, both of those properties were strongly affected by [Na+]p. At fixed depolarizing potentials, the outward current increased approximately as the square power of [Na+]p, a feature that cannot be easily explained by one-ion models for a channel or by "continuum" theories based on electrodiffusion. Instead, all the data could be well described by a "single-file" model for a two-site pore that admits up to two ions. Although double occupancy of the Ca channel by divalent cations has been proposed previously (Hess and Tsien. 1984. Nature. 309: 453-456; Almers et al., 1984. J. Physiol. 353: 585-608), this study indicates that, in our system, states of the channel with two Na+ ions must also be considered in order to explain the dependence of the outward current on [Na+]p. A good fit to the data could be obtained by assuming that both sites in the channel are "electrically" close to its cytoplasmic end and that most of the voltage dependence pertains to the rates for ion exit to the external medium. The values of the parameters suggest that: (a) Ca2+ is bound most strongly by the site nearest to the cytoplasm (in both singly and doubly occupied channels); (b) in channels with two Ca2+ ions, the dissociation constant of the site close to the external mouth must be greater than 2.5 mM; and (c) in pores occupied by two Na+ ions, the rate constant for Na+ exit to the external solution is larger than the rate constant for Na+ exit to the cytoplasm.