The role of anion and cation channels in volume regulatory responses in trout red blood cells

(1) An outwardly rectifying chloride channel (ORCC) of large conductance ha s been detected under isotonic conditions (320 mosM l(-1)) in the plasma me mbrane of trout red blood cells (RBCs) using the excised inside-out configu ration. The channel, with a permeability ratio P-Cl/P-cation of 12, was inh ibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) (50 muM). and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DID S) (100 muM) in the bathing solution. (2) In hypotonic conditions (215 mosM l(-1)), 44% of cell-attached patches showed spontaneous single channel activity identified as nonselective catio nic (NSC) channels. A second group, corresponding to 7% of cell-attached pa tches, showed spontaneous activity corresponding to a channel type presenti ng outward rectification and anionic selectivity. Finally, 49% of patches d isplayed a complex spontaneous signal corresponding to the superimposition of inward and outward currents probably due to activation of different chan nel types. (3) Giga-seals obtained without suction in intact cells under isotonic cond itions possessed NSC channels that were quiescent but which could be activa ted either by mechanical deformation of cell membrane or by hypotonic cell swelling. (4) Hypotonically swollen RBCs exhibited regulatory volume decrease (RVD) o ver 3 h, which was linked to a fivefold to sixfold increase in unidirection al fluxes of K+, a net loss of intracellular Kf and net gain of extracellul ar Na+. RVD and the hypotonically activated, unidirectional K+ influx conti nued after replacement of Cl- by methylsulfonate (MeSF) albeit more slowly. (5) The NSC channel inhibitor, barium, and the Cl- channel inhibitor, NPPB, both inhibited the RVD response by similar to 50% in Cl- containing saline . When Cl- was replaced by MeSF, the inhibition was >90% suggesting that NS C channels and ORCC play key roles in the chloride-independent component of RVD. (C) 2000 Elsevier Science S.A. All rights reserved.