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Use of a purified and functional recombinant calcium-channel β4 subunit in surface-plasmon resonance studies

Abstract : Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Cav and at least two auxiliary subunits α2δ and β. The β subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native β subunit. Here, we report the purification of a recombinant calcium-channel β4 subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Cav-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the β4 subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-β4 subunit for the full-length Cav2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Cav/β interaction and (ii) crystallographic studies of the calcium-channel β4 subunit.
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https://hal-amu.archives-ouvertes.fr/hal-03263160
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Submitted on : Thursday, June 17, 2021 - 8:56:48 AM
Last modification on : Friday, July 2, 2021 - 8:44:03 AM

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Sandrine Geib, Guillaume Sandoz, Kamel Mabrouk, Alessandra Matavel, P. Marchot, et al.. Use of a purified and functional recombinant calcium-channel β4 subunit in surface-plasmon resonance studies. Biochemical Journal, Portland Press, 2002, 364 (1), pp.285-292. ⟨10.1042/bj3640285⟩. ⟨hal-03263160⟩

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