Control of presynaptic function by a persistent Na(+) current

Abstract

Little is known about ion channels that regulate the graded, subthreshold properties of nerve terminals. Using the calyx of Held, we demonstrate here a large presynaptic persistent Na(+) current with unusually hyperpolarized activation voltage. This feature allowed the current to determine both the resting potential and resting conductance of the nerve terminal. Calyces express presynaptic glycine receptors whose activation depolarizes the synapse. We found that activation of the persistent Na(+) current was an essential component in the response to glycine. This Na(+) current originated at or very close to the terminal and was sustained even after trains of large spike-like depolarizations. Because Na(+) channels also underlie the presynaptic action potential, we conclude that their action both triggers and modulates exocytosis through control of presynaptic membrane voltage.

Fig 1

Activation of a presynaptic I_NaP. (A) A depolarizing voltage step evoked a transient Na⁺ component (arrow) followed by a steady-state (persistent) current (black trace). Both components were fully blocked by 500 nM TTX (red trace). The inset shows the I_NaP at higher gain. Leak subtraction was applied to the traces. (B) Influence of ramp speed on I_NaP. The ramp speeds are +280, 70 and 16 mV/s for red, green and blue traces, respectively. The current is partially blocked by 10 nM TTX (grey trace) and fully blocked by 500 nM TTX (black trace). Data in A and B were obtained from one calyx. (C) Expanded view of activation of current in b for two ramps speeds after subtracting trace in TTX. Activation of current is first apparent at about −85 mV. (D) Conductance voltage curve for one calyx, with representative voltage steps shown as inset. Red line is Boltzmann fit with parameters as indicated. (E) Effect of conditioning “spikes” on I_NaP. Left panel is control response to a voltage step to −40 mV. Peak current is cut off. Right panel shows a step to −40 mV preceded by 40 1-ms steps to +20 mV delivered at 200 Hz. A 4-ms return to −80 mV followed the last brief pulse and before the test pulse to −40 mV. After the pulse train, the mean I_NaP measured 250–300 ms after pulse onset was 91.2 ±1.8% of control (n=5).

Fig. 2

Effects of TTX on resting presynaptic membrane properties. (A–B) Puff application of 2 μM TTX hyperpolarizes the resting potential by about 2 mV (n=9). (C) Bath application of TTX decreased resting conductance (n=5). Conductance was estimated in current clamp around the resting potential.

Fig 3

Modulation of glycine responses by persistent Na⁺ current. (A) Calyceal membrane potentials were adjusted by currents injection. Puff application of glycine (1 mM at bar) evoked depolarizing responses (black trace). Bath applied TTX (red trace) decreased the glycine response at voltages between −75 mV and −90 mV, but had no effect at −60 mV and −105 mV. (B) Average data show a maximal effect of TTX on glycine responses (black, control; red, TTX) evoked near the resting potential (symbol at bottom of figure). p < 0.05 for −70 and −90 mV; p < 0.01 for −80 mV, n=5. (C) At −80 mV, close to average resting potential, the glycine response was significantly decreased by TTX (p < 0.001, n=8). (D) Under voltage clamp, the glycine-induced current was not affected by application of TTX in one representative calyx. (E) Data from 7 voltage-clamped calyces showing that TTX does not alter the glycine-evoked current (V_HOLD=−80mV).

Fig 4

Na⁺ channels augment the glycine effect on mEPSC frequency. Glutamatergic spontaneous EPSCs are recorded under control condition (A), 200 μM glycine (B), and 200 μM glycine plus 500 nM TTX (C). V_HOLD = −77 mV. (D) Relative change of spontaneous frequency by glycine (gly) and glycine plus TTX. The frequency in the presence of glycine is significantly decreased by TTX.