Although N- and P-type Ca2+ channels predominant in fast-secreting systems Lc-type

Although N- and P-type Ca2+ channels predominant in fast-secreting systems Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. coexpressed in oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65 where at a certain ratio of p65/syntaxin the channel regains its unaltered kinetic parameters. The cytosolic region of the channel Lc753–893 separating repeats II–III of its α1C subunit interacts with p65 and “pulls” down native p65 from rat brain membranes. Lc753–893 injected into single insulin-secreting β-cell inhibits secretion in response to channel opening but not in response to photolysis of caged Ca2+ nor does it affect Ca2+ current. These results suggest that Lc753–893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis. oocytes expression system. Third the consequence of these interactions was investigated by capacitance measurements of insulin release in Arry-520 single β-cells injected with the channel-interacting domain. Our results provide a molecular and functional basis to suggest the formation of a multiprotein complex (excitosome) composed of the Lc channel p65 and soluble Arry-520 N-ethylmaleimide-sensitive attachment proteins receptors which allows temporal and spatial coupling of Arry-520 the channel to the exocytotic vesicles. MATERIALS AND METHODS Constructs of Glutathione (3088) fragment into the site in QE 30 (Qiagen). The basepair numbers were according to GenBank accession no. “type”:”entrez-nucleotide” attrs :”text”:”M67516″ term_id :”206575″ CTSL1 term_text :”M67516″M67516 (rat brain calcium channel α-1 subunit). GST fusion proteins of SNAP-25 syntaxin p65(1–5) p65(1–3) and p65(3–5) were gifts (see BL21pLysS (Novagen). Purification of His6-Tagged Fusion Proteins. Bacterial pellets were thawed in the presence of 8 M urea 150 mM NaCl 50 mM Tris?HCl (pH 8.0) and 1% Triton X-100 and applied to nickel-nitrilotriacetic acid agarose beads. The beads were subjected to a decreasing gradient of urea 8 M and washed with 150 mM NaCl 50 mM Mes buffer pH 6.0. After elution with 0.3 M imidazole (pH 7.0) the eluate was dialyzed against PBS. The purified protein Arry-520 was detected by Coomassie blue and Western analysis by using affinity-purified antibodies raised against the L-peptide TTKINMDDLQPSENEDKS and the N-peptide RHHRHRDRDKTSAST (9). Oocytes. Stage V–VI oocytes were removed surgically from the ovaries of anesthetized animals and transferred to a Ca2+-free ND96 solution (mM): 96 NaCl 2 KCl 1 MgCl2 5 Hepes (pH 7.4) containing 2 mg/ml collagenase (154 units/mg Worthington). The follicular cell layer was removed and the oocytes were then washed extensively and placed into a ND96 solution containing 1.8 CaCl2 2.5 mM sodium pyruvate 100 units/ml penicillin and 100 μg/ml streptomycin at 20°C for 12–20 h before cRNA injection. Arry-520 Plasmid DNA for the channel subunits α*1C α2δ β2A syntaxin 1A SNAP-25 and p65 (8) were linearized treated with proteinase K and transcribed with T7 polymerase and transcription kit (Stratagene) in the presence of the cap analog G (5′) ppp(5′)G (Pharmacia). The Oocytes. Whole cell currents were recorded by applying a standard two-microelectrode voltage clamp by using a Dagan 8500 amplifier (Dagan Instruments Minneapolis) (8). Voltage and current agar-cushioned electrodes (0.3- Arry-520 to 0.6-mΩ tip resistance) were filled with 3 M KCl. Current-voltage relationships were determined by voltage steps from ?80 to +60 mV of 500 ms duration with 30 sec intervals in Ba2+ solution (in mM): 40 Ba(OH)2 50 N-methyl d-glucamine 1 KOH and 5 Hepes (pH 7.5) titrated to pH 7.5 [(CH3)2SO4]. Voltage-dependent inactivation was carried out as described (8) and fitted by a single Boltzmann distribution with normalized current = C/{1 + exp[(the slope parameter. Activation kinetics were determined by using 100 ms leak subtracted pulses to +20 mV. Current traces were analyzed.