Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.
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.
Geib
Sandrine
S
INSERM EMI 99-31, Canaux Ioniques et Signalisation, CEA, DBMS, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France.
Sandoz
Guillaume
G
Mabrouk
Kamel
K
Matavel
Alessandra
A
Marchot
Pascale
P
Hoshi
Toshinori
T
Villaz
Michel
M
Ronjat
Michel
M
Miquelis
Raymond
R
Lévêque
Christian
C
de Waard
Michel
M
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
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