title: Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.
abstract: Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta 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 beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) 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 Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) 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-beta(4) subunit for the full-length Ca(v)2.1 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 Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit.
authors: INSERM EMI 99-31, Canaux Ioniques et Signalisation, CEA, DBMS, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France.
authors: Sandrine
authors: S
authors: Geib
authors: Guillaume
authors: G
authors: Sandoz
authors: Kamel
authors: K
authors: Mabrouk
authors: Alessandra
authors: A
authors: Matavel
authors: Pascale
authors: P
authors: Marchot
authors: Toshinori
authors: T
authors: Hoshi
authors: Michel
authors: M
authors: Villaz
authors: Michel
authors: M
authors: Ronjat
authors: Raymond
authors: R
authors: Miquelis
authors: Christian
authors: C
authors: Lévêque
authors: Michel
authors: M
authors: de Waard
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels / Q000737:chemistry* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007477:Ions; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors; D014982:Xenopus laevis
chemical_list: C491529:Cacnb4 protein, rat; D015220:Calcium Channels; D018076:DNA, Complementary; D007477:Ions; D010455:Peptides; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D005982:Glutathione Transferase