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J. Biol. Chem., Vol. 279, Issue 21, 21689-21694, May 21, 2004

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Folding of Active Calcium Channel _1b -Subunit by Size-exclusion Chromatography and Its Role on Channel Function^*

Alan Neely, Jennie Garcia-Olivares¶, Stephan Voswinkel, Hannelore Horstkott, and Patricia Hidalgo||**

From the Centro de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso 2349400, Chile, Institut für Physiologie, Rheinisch-Westfälische Hochschule Aachen, 52074 Aachen, Germany, and ^||Centro de Estudios Científicos, Valdivia 509000, Chile

Voltage-gated calcium channels mediate the influx of Ca²⁺ ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming ₁-subunit and the auxiliary - and ₂-subunits. The -subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four -subunit genes have been cloned, _1–4, with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The _1b-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming ₁- and the regulatory -subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the _1b-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The _1b-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the ₁-subunit (the -interaction domain). Using the cutopen oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac ₁-subunit (_1C) co-injected with folded-_1b-protein or _1b-cRNA. We demonstrate that the co-expression of the _1C-subunit with either folded-_1b-protein or _1b-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the _1b-subunit primarily modulates channel activity, rather than expression.

Received for publication, November 19, 2003 , and in revised form, March 10, 2004.

^* This work was supported by Fondo Nacional de Desarrollo Cientí-fico y Tecnológico Grants 1020899, ICM P99-037-F (to A. N.), and Deutsche Forschungsgemeinschaft Grants FOR 450 and TP 1 (to P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Student of Programa de Doctorado, Facultad de Ciencias de la Universidad de Chile and Universidad de Valparaíso.

^** To whom correspondence should be addressed: Institut für Physiologie, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. Tel.: 49-24180-88810 or -88873; Fax: 49-24180-82434; E-mail: patty{at}physiology.rwth-aachen.de.

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