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Chloride and non-selective cation channels in unstimulated trout red blood cells

Stéphane Egée, Olivier Mignen, Brian J. Harvey * and Serge Thomas

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were used to demonstrate the presence of two different types of ion channels in the membrane of trout red blood cells under isotonic and normoxic conditions, in the absence of hormonal stimulation. The large majority (93 %) of successful membrane seals allowed observation of at least one channel type.

2. In the cell-attached mode with Ringer solution in the bath and Ringer solution, 145 mM KCl 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 single channel activity reversed between 5 and 7 mV positive to the spontaneous membrane potential. A small conductance channel of 5-6 pS and +5 mV reversal potential was also present in 62 % of cells.

3. After excision 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 intermediate conductance channel was present in 439 out of 452 successful seals. This channel was spontaneously active in 90 % of patches and in the other 10 % of patches the channel was activated by suction. The current-voltage relationship showed slight inward rectification. The channel conductance 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 zero. Substitution of K⁺ for Na⁺ in the pipette or in the bath did not significantly change the single channel conductance. Dilution of the bathing solution KCl concentration shifted the reversal potential towards the Nernst equilibrium for cations. Substitution of N-methyl-D-glucamine (NMDG) for K⁺ or Na⁺ in the bath almost abolished the outward current whilst the divalent cation Ca²⁺ permeated the channel with a higher permeability than K⁺ and Na⁺. Inhibition of channel openings was obtained with flufenamic acid, quinine, gadolinium or barium. Taken together these data demonstrate that the intermediate conductance channel belongs to a class of non-selective cation (NSC) channels.

4. In excised patches, under the same control conditions, the conductance of the small conductance non-rectifying channel was 8·6 ± 0·8 pS (n = 12) between -60 and +60 mV and the reversal potential was close to 0 mV. This channel could be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) but not by flufenamic acid, DIDS, barium or gadolinium. Selectivity and substitution experiments made it possible to identify this channel as a non-rectifying small conductance chloride (SCC) channel.

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