Folding of Active Calcium Channel beta sub(1b)-Subunit by Size-exclusion Chromatography and Its Role on Channel Function

• Published in: The Journal of biological chemistry 2004-05, Vol.279 (21), p.21689-21694
• Main Authors: Neely, A, Garcia-Olivares, J, Voswinkel, S, Horstkott, H, Hidalgo, P
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M312675200

Voltage-gated calcium channels mediate the influx of Ca super(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha sub(1)-subunit and the auxiliary beta - and alpha sub(2) delta -subunits. The beta -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 beta -subunit genes have been cloned, beta sub(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta sub(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha sub(1)- and the regulatory beta -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 beta sub(1b)- subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta sub(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 alpha sub(1)-subunit (the alpha -interaction domain). Using the cutopen oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha sub(1)-subunit ( alpha sub(1C)) co-injected with folded- beta sub(1b)-protein or beta sub(1b)-cRNA. We demonstrate that the co-expression of the alpha sub(1C)-subunit with either folded- beta sub(1b)-protein or beta sub(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta sub(1b)-subunit primarily modulates channel activity, rather than expression.