Modification of C1- transport in skeletal muscle of Rana temporaria with the arginine-binding reagent phenylglyoxal

Abstract

1. The effect of membrane modification by the arginine-binding reagent phenylglyoxal (PG) on Cl- permeability was studied in thin bundles of twitch fibres from frog muscle. The bundles were modified by a method that yields stable PG binding to outer arginyl residues in erythrocyte membranes. 2. PG almost eliminated the pH-dependent fraction of 36Cl- efflux under conditions of Cl- equilibrium in depolarized bundles: the fluxes at pH 7.2 and 8.5 were strongly inhibited approaching an apparent baseline value close to the normal flux at pH 6 which per se was not inhibited. 3. The uninhibited flux at pH 6 in modified bundles maintained the normally high sensitivity to 4,4'-dinitro-stilbene-2,2'-disulphonate (DNDS), and the reduction of fluxes at pH > 7 coincided with increased DNDS sensitivity, suggesting a selective blocking of the pH-dependent flux fraction that has a low DNDS sensitivity. 4. In normal Ringer solution the modified fibres showed normal resting membrane potentials (Vm) with normal sensitivity to [K+]o but sensitivity to changes of [Cl-]o was almost eliminated, suggesting a normal resting Na+:K+ conductance ratio (gNa/gK) and that the main influence of modification on the resting membrane conductance (gm) was a loss of Cl- conductance (gCl). 5. The modified fibres were not excitable, possibly due to arginine modification in the voltage sensor (S4) of the Na+ channels. 6. These results suggest that positively charged arginines are important for the activity of the pH-dependent Vm-stabilizing Cl- channels and that PG may isolate a pH-independent basal flux fraction which normally dominates the Cl- flux at low pH.

Figure 6. Example of ³⁶Cl⁻ efflux as a function of time in an irreversibly modified bundle compared with efflux in a control bundle from the same animal

Logarithmic ordinate. The set of experiments was made on frog No. 12 in Table 2. The modified bundle was exposed to 20 mM PG at 0 mM Cl⁻ and 20 °C in 60 s before incubation at 0 °C. Note that the pH dependence of efflux was almost eliminated in the modified bundle, while the reversible fractional inhibition with DNDS (5 × l0⁻⁴ mM) at pH 6 was about the same in both bundles (cf. below: ‘Inhibition of the residual flux with the anion transport inhibitor DNDS’).

Figure 5. Mean ³⁶Cl⁻ efflux rate coefficients in untreated control bundles and bundles that were irreversibly modified with exposure to 20 mM PG (45–75 s) at low [Cl⁻]

Bars show the standard deviations based on the following number of observations (control and PG modification, respectively): pH 8.5, 6 and 8; pH 7.2, 11 and 12; pH 6, 5 and 5.

Figure 1. Inhibition of ³⁶Cl⁻ efflux at pH 7.2 by exposure to phenylglyoxal (PG) during ³⁶Cl⁻ washout from depolarized fibre bundles

Ordinate: efflux rate coefficient relative to that before exposure to PG. Arrows point to the symbols indicating the mean efflux rate during exposure to PG (10 mM PG, 1 min). In one bundle (^) exposure to PG was carried out at pH 7.2. In the other bundle (•), exposure to PG was carried out at pH 10. The superfusion at pH 10 was initiated 2 min before PG treament, as revealed by the increase of efflux rate.

Figure 2. ³⁶Cl⁻ efflux at pH 7.2 with three consecutive exposures to PG at pH 10

Ordinate as in Fig. 1. •, efflux during the exposures to PG (10 mM PG for 1 min). ^, control. Note the simultaneously occurring reduction in efflux responses to alkalization and to exposure to PG.

Figure 3. Effects of repeated exposure to PG on ³⁶Cl⁻ efflux at pH 6 and 10

Ordinate: rate coefficient relative to that during the initial efflux at pH 7.2. •, exposure to PG (10 mM) for 1 min at pH 10. Note the almost complete absence of an inhibitory effect at pH 6, and that the residual flux rate at pH 10 after the last exposure to PG is similar to that at pH 6.

Figure 4. The immediate effect of exposure to PG on Cl⁻ net efflux at pH 7.2

The figure shows experiments on five fibre bundles, three of which were exposed to PG (10 mM, pH 10 for 1 min) at the 10th minute. •, efflux rates from all experiments before PG treatment and after the 9th minute from two control bundles. ^, efflux rates from PG-treated bundles. Ordinate: efflux rate coefficient relative to that at chloride equilibrium (before time zero). Bars indicate s.d. (or range when only two observations are included: • after the 10th min). No bars mean that the s.d. lies within the symbol except for the last two observations of both treated and untreated bundles belonging to one extended set of experiments on two bundles from the same animal. All bundles were uniformly treated until the time of exposure to PG (• before the 10th min): (i) depolarization and ³⁶Cl⁻ loading for 2–3 h at pH 7.2 in 140 mM K⁺ and 20 mM Cl⁻ (solution 3, Table 1); (ii) ³⁶Cl⁻ washout at Cl⁻ equilibrium by superfusion with radioactive-free solution 3 at pH 7.2 (initial flux rate before time zero); (iii) establishment of ³⁶Cl⁻ net efflux (time zero) by a change to Cl⁻-free superfusion solution at pH 7.2 (solution 4, Table 1); (iv) the temporary change to pH 10 before exposure to PG. Note the missing flux activation in PG-treated bundles upon the change of pH from 10 to 7.2 (^).

Figure 8. The influence of irreversible modification on the sensitivity of ³⁶Cl⁻ efflux to DNDS

In each of the three examples the fractional inhibition of ³⁶Cl⁻ efflux with DNDS is shown for irreversibly modified (•) and unmodified (^) depolarized bundles from the same animal. Arrows indicate the initiation of DNDS exposure. A, inhibition at pH 6 with 1.25 mM DNDS (No. 14, Table 2). B, inhibition with 5 × 10⁻⁴ M DNDS at pH 6 (No. 12, Table 2) and pH 7.2 (No. 16, Table 2).

Figure 7. Example of the influence of [Cl⁻]_o during exposure to PG on the degree of irreversible modification

The set of experiments was made on fibre bundles from the same animal (No. 17, Table 2, included in Table 3B). Upon phenylglyoxalation (20 mM PG during 70 s) and incubation at 0 °C for >20 h, both bundles were depolarized, loaded with ³⁶Cl⁻ (4 h), and washed in solution 3 (Table 1) at various pH values. The dashed lines represent the mean rate coefficients in unmodified bundles at pH 6 and 7.2 (identical with controls in Fig. 5). At pH 8.5 the mean rate coefficient in unmodified bundles was 0.23 min⁻¹. Note the attenuated inhibition by PG treatment at 122 mM Cl⁻ compared with treatment at 0 mM Cl⁻.

Figure 9. The influence of irreversible modification on the resting membrane potential and its sensitivity to changes of [Cl⁻]_o and [K⁺]_o

Modified and control fibres were from the same animal. ‘i’ indicates insertion of microelectrode. ‘o’ indicates withdrawal of microelectrode. When not indicated, the medium was normal frog Ringer solution (solution 1, Table 1). 0 mM Cl⁻: sulphate Ringer solution (solution 2b). 30 mM Cl⁻ (solution 5): the solution used by Hodgkin & Horowicz (1959, Fig. 2). 15 mM K⁺: 13 mM KCl was added to solution 1. A, recordings from five (a-e) different superficial fibres of a PG-treated bundle. Compared with normal fibres (B), the figure shows (i) normal values of V_m (-80 to −90 mV), (ii) reversed V_m response to a change from 120 to 30 mM Cl⁻ (a and b) and only a small depolarization upon the changes to 0 mM Cl⁻ (c and e), and (iii) a normal response to increased [K⁺]_o (d and e). B, recordings from two (a and b) normal fibres. The traces are simultaneous recordings at different amplifications. The lower trace at low amplification shows the measured potential without the corrections of the trace level needed for fibre b shown in the upper trace in B at higher magnification. The record from fibre b shows fractions of one impalement lasting 80 min.

Figure 10. The resting membrane potential (V_m) as a function of [K⁺]_o in superficial fibres of an irreversibly modified bundle

The solutions used (mixtures of solutions 6 and 7, Table 1) were the Cl⁻-free solutions used for the same purpose by Hodgkin & Horowicz (1959, Fig. 5). Continuous line represents the equilibrium potential of [K⁺], assuming an internal [K⁺] of 140 mM. The measurements were first made during increasing [K⁺]_o (^), then during decreasing [K⁺]_o (□), and finally V_m was tested at 10 mM ${\text{K}}_{\text{o}}^{+}$ (▿). When the measured potential drifted, a new fibre in the bundle was impaled.