Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones

Abstract

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non-specific ion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non-specific ion channel was examined using the fused vesicle-attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

Figure 1. The pH dependence of the opening of the non-specific vesicle channel

A, the mean frequency of channel opening (mean ±s.d.): 2.9 ± 1.4 Hz, pH 5.5; 8.19 ± 2.6 Hz, pH 6.0; 69.5 ± 9.7 Hz, pH 6.5; 757.9 ± 69.5 Hz, pH 7.25; 1 s bins. B, the mean channel closed time (ms) decreases with the elevation of pH (mean ±s.e.m.): 1456.87 ± 95.8, pH 5.5; 691.66 ± 130.76, pH 6.0; 62.07 ± 9.06, pH 6.5; 0.476 ± 0.12, pH 7.25.

Figure 2. Open probability of the non-specific ion channel as a function of voltage at four different pH values

The open probability (NP_o) decreases with acidification. In each pH it reaches the highest value at 0 mV and decreases fast upon imposing voltage on the membrane. A, pH 5.5; B, pH 6.0; C, pH 6.5; D, pH 7.25. E, 3D presentation of the probability of opening as a function of both voltage and pH. The error bars represent standard errors of the mean.

Figure 3. Control experiments

A–D, i–V relation at different pH values. A, pH 5.5, slope conductance = 173.36 pS, intercept = 15.0 mV; r² = 0.99; n = 7. B, pH 6.0, slope conductance = 181.77 pS, intercept = 17.7 mV; r² = 0.97; n = 8. C, pH 6.5, slope conductance = 165.80 pS, intercept = 16.4 mV; r² = 0.97; n = 5. D, pH 7.25, slope conductance = 162.78 pS, intercept = 17.0 mV; r² = 0.99; n = 3. The error bars represent standard deviations. E, mean channel open time does not depend significantly on pH. Student's t test yielded P-values greater than 0.05 for most of the slopes indicating that there is no significant difference in the slope conductance at the examined pH values.