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

Abstract

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, Vp = 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.

Figure 1. Non-selective cation (NSC) channel in intact cells

A, representative tracings from single-channel currents of non-selective cation (NSC) channel in cell-attached patches at the indicated holding potentials. The bath contained isotonic Ringer solution and the pipettes were filled with KCl solution. The closed state is shown by the dashed line. Upward deflection at positive clamp potentials indicate the flow of cations from pipette to cell interior. B, current-voltage relationship. •, under similar conditions to that of the upper traces; bath, Ringer solution; pipette, KCl solution; n= 24 experiments; ○, after replacement of KCl by potassium gluconate; bath, Ringer solution; pipette, K-gluconate solution; n= 5.

Figure 2. Small conductance chloride (SCC) channel in intact cells

A, cell-attached recordings showing the presence of one or two small conductance chloride (SCC) channel together with the NSC channel. B, current-voltage relationship of SCC channel from 8 experiments with isotonic Ringer solution in the bath and KCl solution in the pipette.

Figure 3. Current-voltage relationship corresponding to the data contained in Table 2 of the NSC channel

Current-voltage relationship corresponding to the data contained in Table 2 of the NSC channel in excised inside-out patches at different V_m. A, bath, K_int solution; •, pipette, KCl solution; n= 35 experiments; ○, pipette, NaCl solution; n= 8; □, pipette, K-gluconate solution; n= 12. B, bath, Na_int solution; •, pipette, KCl solution; n= 19; ○, pipette, NaCl solution; n= 3.

Figure 4. Current-voltage relationships of substitution or selectivity experiments

Current-voltage relationships of substitution or selectivity experiments and constructed from recordings where K_int was replaced by K_int,½ (A) or by NMDG (B) in the bath, or where KCl was replaced by 72.5 mm CaCl₂ in the pipette (B). A, •, bath, K_int solution; pipette, KCl solution; n= 35 experients; ○, bath, K_int,½ solution; pipette, KCl solution; n= 10. B, •, bath, NMDG solution; pipette, KCl solution; n= 17; ○, bath, K_int solution; pipette, CaCl₂ solution; n= 13; □, bath, Na_int solution; pipette, CaCl₂ solution; n= 6.

Figure 5. Open probability of the NSC channel

A, P_o-V_m plot showing that the open probability (P_o) of the NSC channels was not related to the membrane potential (V_m) in the single channel current recordings when the inside-out patches exhibited spontaneous NSC channel activity after excision (90 % of cases). B, when the NSC channel activity was induced by suction (10 % of cases), the P_o value, measured at a membrane potential of -20 mV was clearly related to the intensity of the depression imposed on the membrane patch (n= 12).

Figure 6. NSC channel inhibition by gadolinium

A, typical example of the progressive reduction of P_o and number of channels N induced by gadolinium (20 μM) in the patch pipette. B, representative tracings from single channel currents of the NSC channel in excised inside-out patches at V_m= -30 mV, before or immediately after addition of 5 mm Ba²⁺ in the bathing solution. The bath contained K_int solution and the pipettes were filled with KCl solution.

Figure 7. Single-channel current-voltage relationships of the SCC channel recorded from excised inside-out patches

•, bath, K_int solution; pipette, KCl solution; n= 12; voltage commands between -60 and -30 mV and between +30 and +60 mV. Under these symetrical conditions the reversal potential was close to +5 mV. ▵, bath, K_int solution; pipette, K-gluconate solution; n= 12. The positive currents disappeared when gluconate replaced chloride in the pipette-filling solution. ○, bath, K_int,½ solution; pipette, KCl solution; n= 5. The I-V relationship was shifted to the left when the bathing solution was K_int,½.