Table 1.
Primers used to identify Cav channels α subunits in SH-SY5Y cells.
Figure 1.
RT-PCR to identify the Cavα and auxiliary subunit isoforms expressed in SH-SY5Y cells.
Expression of Cavα subtypes, auxiliary β, α2δ and γ subunits, as well as Cav2.2 splice variant isoforms were determined in SH-SY5Y cells using standard RT-PCR and specific primers for each isoform. (A) SH-SY5Y cells endogenously express Cav1.3 isoform 1, Cav1.3 isoform 2, Cav2.2 and Cav3.1, but not Cav1.1 and Cav1.2, Cav1.4, Cav2.3, Cav3.2 and Cav3.3. Expected band sizes were (bp): Cav1.3 isoform 1, 541; Cav1.3 isoform 2, 343; Cav2.2, 754; and Cav3.1, 397, as indicated with arrows (B) SH-SY5Y cells endogenously express different Cav2.2, α1B splice variant isoforms. Bands with predicted sizes were (bp): α1B1, 728; α1B2, 854; Δ1, 900 bp. No band was detected for splice Δ2. (C–D) SH-SY5Y cells express the auxiliary β1, β3, and β4 but not β2; in addition to α2δ1–3, but not α2δ4; and γ1, γ4–5 and γ7 but not γ2–3 and γ8 subunits. Expected band sizes were (bp, base pairs): β1, 331; β3, 594; β4, 731; α2δ1, 252; α2δ2, 878; α2δ3, 132 and γ1, 367; γ4, 909; γ5, 257; and γ7, 910.
Table 2.
Primers used to identify Cav channel auxiliary subunits in SH-SY5Y cells.
Figure 2.
Displacement of 125I-GVIA from SH-SY5Y whole cell and membranes byω-conotoxins.
Displacement of 125I-GVIA binding to Cav2.2 expressed in rat brain and SH-SY5Y intact/whole cell and membranes. (A) Displacement of 125I-GVIA from rat brain membranes. (B) Displacement of 125I-GVIA from human SH-SY5Y cell membranes. (C) Displacement of 125I-GVIA from human SH-SY5Y whole cell. (D) ω-Conotoxins affinity (Kd ± SEM) to displace 125I-GVIA from rat brain membranes and human SH-SY5Y cell membranes. Data are mean ± SEM of triplicate data from a representative experiment best fitted to a single-site competition model using GraphPad Prism.
Table 3.
ω-Conotoxin affinities (IC50± SEM) to displace 125I-GVIA binding.
Figure 3.
Cav2.2 and Cav1 channels endogenously expressed in SH-SY5Y cells are functional.
Data obtained from fluorescent Ca2+ imaging assays of KCl-evoked Ca2+ responses in SH-SY5Y cells. (A) Cav1 and Cav2.2 activation in the presence of CVID (open ball) and nifedipine (filled ball), respectively, shifted control KCl-evoked Ca2+ responses (quadrilateral) significantly in SH-SY5Y cells (p>0.05). (B) Time course of Ca2+ responses is shown for control KCl 90 mM (black), KCl in the presence of nifedipine (blue) and KCl in the presence of CVID (green). (C) Concentration-response curve for nifedipine inhibition of Cav1 responses (D) Concentration-response curves for CVID, GVIA and MVIIA inhibition of Cav2.2 responses. The responses were normalized using controls: positive KCl and negative PSS buffer; and plotted across increasing concentrations of antagonists (E) Comparison of ω-conotoxins CVID, GVIA and MVIIA potencies (IC50/Kd ± SEM of n = 3–4 replicates for each experiment, n = 3 experiments) in displacing 125I-GVIA from SH-SY5Y whole cell and SH-SY5Y cell membranes with the functional assays data.
Table 4.
Potency (IC50± SEM) of Cav channel modulators on functional assays.
Figure 4.
Characterization of resistant Ca2+ responses in SH-SY5Y cells.
Data obtained from fluorescent Ca2+ imaging of KCl-evoked Ca2+ responses in SH-SY5Y cells. (A) Concentration-response curves for mibefradil, pimozide, ω-agatoxin TK and SNX 482 in inhibiting resistant KCl-evoked Ca2+ responses in SH-SY5Y cells, pretreated with CVID (3 µM) plus nifedipine (10 µM) (B–D) Time course of transient Ca2+ responses activated by 90 mM KCl/5 mM CaCl2, in the presence of CVID (3 µM) and nifedipine (10 µM) and following the addition of agatoxin TK, SNX-482 and mibefradil.