CHLORIDE AND NONSELECTIVE CATION CHANNELS IN UNSTIMULATED TROUT RED-BLOOD-CELLS

1. The cell-attached and excised inside-out configurations of the patc h-clamp technique were used to demonstrate the presence of two differe nt types of ion channels in the membrane of trout red blood cells unde r isotonic and normoxic conditions, in the absence of hormonal stimula tion. The large majority (93%) of successful membrane seals allowed ob servation of at least one channel type. 2. In the cell-attached mode w ith Ringer solution in the bath and Ringer solution, 145 mM KCI or 145 NaCl in the pipette, a channel of intermediate conductance (15-25 pS at clamped voltage, V-p = 0 mV) was present in 85% of cells. The singl e channel activity reversed between 5 and 7 mV positive to the spontan eous membrane potential. A small conductance channel of 5-6 pS and +5 mV reversal potential was also present in 62% of cells. 3. After excis ion into the inside-out configuration (with 145 mM KCl or NaCl, pCa 8 in the bath, 145 mM KCl or NaCl, pCa 3 in the pipette) the intermediat e conductance channel was present in 439 out of 452 successful seals. This channel was spontaneously active in 90% of patches and in the oth er 10% of patches the channel was activated by suction. The current-vo ltage relationship showed slight inward rectification. The channel con ductance was in the range 15-20 pS between -60 and 0 mV and increased to 25-30 pS between 0 and 60 mV, with a reversal potential close to ze ro. Substitution of K+ for Na+ in the pipette or in the bath did not s ignificantly change the single channel conductance. Dilution of the ba thing solution KCl concentration shifted the reversal potential toward s the Nernst equilibrium for cations. Substitution of N-methyl-D-gluca mine (NMDG) for K+ or Na+ in the bath almost abolished the outward cur rent whilst the divalent cation Ca2+ permeated the channel with a high er permeability than K+ and Na+. Inhibition of channel openings was ob tained with flufenamic acid, quinine, gadolinium or barium. Taken toge ther these data demonstrate that the intermediate conductance channel belongs to a class of non-selective cation (NSC) channels. 4. In excis ed patches, under the same control conditions, the conductance of the small conductance non-rectifying channel was 8.8 +/- 0.8 pS (n = 12) b etween -60 and +60 mV and the reversal potential was close to 0 mV. Th is channel could be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoat e (NPPB) but not by flufenamic acid, DIDS, barium or gadolinium. Selec tivity and substitution experiments made it possible to identify this channel as a non-rectifying small conductance chloride (SCC) channel.