Abstract
SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms) and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1–3, β1, β3, β4, γ1, γ4–5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold) higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.
Highlights
Voltage-gated Ca2+ channels (Cav) are membrane proteins essential for the control of calcium signaling events, such as muscle contraction, gene expression, and neurotransmitter and hormone release
Since splice variants can be generated by alternative RNA processing, which can influence function and pharmacology [16], we investigated the expression of some human splice variants [16,17]
Cav2.2 channels play a key role in regulating nociception
Summary
Voltage-gated Ca2+ channels (Cav) are membrane proteins essential for the control of calcium signaling events, such as muscle contraction, gene expression, and neurotransmitter and hormone release. The Cav a subunit contains the voltage sensor and gating machinery and is the binding site for most inhibitors. This subunit comprises 4 domains each with six transmembrane segments. The pore is formed by the S5/S6 segments and the connecting pore loop, with channel opening gated by bending of the S6 segments at a hinge glycine or proline residue [3,4]. The voltage sensor domain consists of the S1–S4 segments, with positively charged residues in S4 serving as gating charges [5] (for review see: [3,6,7])
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