Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function.
Voltage-gated calcium channels mediate the influx of Ca(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(1)-subunit and the auxiliary beta- and alpha(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(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(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(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(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(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.
Neely
Alan
A
Centro de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso 2349400, Chile.
Garcia-Olivares
Jennie
J
Voswinkel
Stephan
S
Horstkott
Hannelore
H
Hidalgo
Patricia
P
D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry; D015220:Calcium Channels / Q000737:chemistry* / Q000502:physiology*; D002138:Calibration; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D007477:Ions; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
D015220:Calcium Channels; D007477:Ions; D011506:Proteins; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D002118:Calcium