Enhancement of presynaptic calcium current by cysteine string protein

Abstract

The isolated chick ciliary neuron calyx synapse preparation was used to test cysteine string protein (CSP) action on presynaptic N-type Ca(2+) channels. Endogenous CSP was localized primarily to secretory vesicle clusters in the presynaptic nerve terminal. Introduction of recombinant CSP into the voltage clamped terminal resulted in a prominent increase in Ca(2+) current amplitude. However, this increase could not be attributed to a change in Ca(2+) channel kinetics, voltage dependence, prepulse inactivation, or G protein inhibition but was attributed to the recruitment of dormant channels. Secretory vesicle associated endogenous CSP may play an important role in enhancing Ca(2+) channel activity at the transmitter release site.

Figure 1. Image slice of a calyx nerve terminal (CX, inset cartoon) attached to a ciliary neuron (CN) and stained for CSP (green) or SV2 (red) with colocalized regions in yellow

Examples of transmitter release face synaptic vesicle clusters are indicated by arrows. Scale bar, 1 μm.

Figure 2. Enhancement of presynaptic calcium current by intracellular CSP

A, current traces from two calyces patched with pipettes containing either plain intracellular solution (inset lower right) or recombinant CSP (inset upper left) and recorded beginning immediately after seal rupture. The voltage protocol was a depolarizing step from −80 to 0 mV. Current amplitudes (I) were measured near the end of the step, normalized to that of the first trial (I₀, trace with dark line) and were plotted (with CSP, ○; control, •). B, currents activated by a depolarizing ramp protocol (top panel) recorded in a control calyx (middle panel) or with CSP (lower panel). Traces were recorded immediately after seal rupture (t = 0, bold) and at 20 (dotted) and 45 s (fine-dotted) thereafter. C, a series of ramp depolarizations were given as in Fig. 2B at 5 s intervals to CSP-infused (□) and vector-control-infused (▪) calyx nerve terminals. The slope conductance, G, was determined as the slope of a straight line fitted to the ascending limb of the current peak between +35 and +40 mV and this was normalized to the value of G at t = 0 (G₀). n = 4 for each treatment.

Figure 3. Effect of CSP on the time course of the Ca²⁺ current

Recordings are from three calyces (A-C) before (thick line) and after (thin line) perfusion through the patch electrode with CSP with voltage protocols above each panel. Raw (leak subtracted) data are shown in the left panel to compare current amplitudes while traces normalized to the current amplitude at the end of the step are on the right to compare current pulse shape. A and B are with a single pulse protocol depolarizing from −80 to 0 mV for 40 ms. In C the test pulse was preceded by a strong (+80 mV, 30 ms) prepulse to remove any voltage-sensitive inhibition.

Figure 4. Effect of intracellular CSP on calcium current inactivation

A, conductance-voltage relation. Current traces were recorded during a ramp depolarization protocol (inset, protocol as in Fig. 2B) before (thick line) and after (thin line) CSP-induced growth. Current traces were corrected for driving force using an E_Ca of +65 mV and were normalized to G_MAX (at ∼+30 mV) and were plotted against the membrane potential to yield G/G_MAX-voltage curves. Curves before (thick line) and after (thin line) CSP action are shown for a single calyx (left panel) and pooled data (right panel). B, prepulse inactivation (PPI) curve. Ca²⁺ current PPI was determined using the voltage protocol in the upper left panel with an interval of 5 s between trials. Traces are shown for control (middle left panel) and CSP-facilitated (lower left panel) calyces. Pooled data normalized to current amplitude with a holding potential of −80 mV from four calyces and fitted Boltzmann relations are plotted before (•, continuous line) and after (○, dashed line) the CSP effect in the right panel.