Neuronal Calcium Sensor 1 and Activity-Dependent Facilitation of P/Q-Type Calcium Currents at Presynaptic Nerve Terminals Tetsuhiro Tsujimoto, Andreas Jeromin, Naoto Saitoh, John C. Roder, and Tomoyuki Takahashi

Supplementary Material

Brainstem slices were prepared from Wister rats (14 - 18 days old) as described (1, 2). The standard aCSF consisted of 120 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, 10 mM glucose, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 3 mM myo-inositol, 2 mM sodium pyruvate, 0.5 mM ascorbic acid, 4 mM lactic acid (pH 7.3 saturated with 95 % O₂ and 5 % CO₂). Experiments were carried out at the room temperature (24 - 26 °C). A slice was transferred to a recording chamber mounted on a stage-fixed upright microscope, and continuously superfused with aCSF at the rate of 1 ml/min. Whole-cell voltage-clamp recordings of presynaptic currents were made from the calyx of Held with patch pipettes pulled from standard wall glass microelectrode having a resistance of 5 - 8 M when filled with an internal solution; 110 mM CsCl, 40 mM HEPES, 0.5 mM EGTA, 12 mM sodium phosphocreatine, 10 mM tetraethyammonium chloride (TEA-Cl), 2 mM ATP, 0.5 mM GTP (pH adjusted to 7.4 with CsOH). To isolate Ca²⁺ currents, TEA-Cl (10 mM) and tetrodotoxin (M) were added to aCSF for blocking voltage dependent Na⁺ and K⁺ currents, respectively. An Axopatch 200B amplifier was used for whole-cell recordings. The series resistance ranged from 7 to 20 M and routinely compensated 80 %. When the uncompensated access resistance exceeded 20 M, data were not adopted. Leakage currents were subtracted by a modified P/N protocol (1, 2). Records were low-pass filtered at 5 KHz by a build-in filter of Axopatch 200B, digitized at 50 KHz, and analyzed using Matlab.

Myristoylated rat NCS-1 was produced in E. coli and purified as described before (3), and dialysed with the presynaptic patch solution.

Before infusion, the presynaptic patch pipette contained 2 l of the patch solution. Ten l of the solution containing NCS-1 (200 M) was infused into the presynaptic terminal through a infusion tube mounted in the presynaptic patch pipette (4) with its tip being placed 150 to 200 m behind the pipette tip.

The half activation voltage (V_1/2) estimated by fitting the I-V curves to with the modified Boltzmann equation, I_pCa = G_max*(V_m - V_eq)/[1 + exp((V_1/2 - V_m)/k)], where G_max, V_m, V_eq, V_1/2 and k represents maximum Ca²⁺ conductance of currents, membrane potential, equilibrium potential of Ca²⁺ current, half activating voltage and slope factor respectively (1, 2).

Fixed brainstem slices (25 m thick) were prepared from 15-day old Wistar rats, and subjected for immunocytochemical examinations as reported (5). The primary antibodies were the rabbit polyclonal anti-NCS-1 (diluted 1:200) (6) and the mouse monoclonal anti-Syntaxin (1:100), the mouse monoclonal anti-MAP2B (1:200) antibodies. For the absorbance experiment, 0.5 l of anti-NCS-1 antibody was incubated with 35 l of 200 M NCS-1 in the pipette solution for 1 h at 37 °C.

The C-terminal peptide (DKNADGKLTLQEFQEGSKADPSIVQALSLYDGLV) and the scrambled peptide (DIDGDGQVNYEEFVQDTLASLSKLAKGLKALPQS) were synthesized according to the solid-phase procedures using standard Fmoc-based chemistry with minor modifications (7). The crude peptides were purified by reverse phase HPLC, collected as more than 99% pure.

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