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 Ca2+ ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the pore-forming ?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 cut-open 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 ?1b-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.
|Título según WOS:||Folding of active calcium channel beta(1b)-subunit by size-exclusion chromatography and its role on channel function|
|Título según SCOPUS:||Folding of active calcium channel ?1b-subunit by size-exclusion chromatography and its role on channel function|
|Título de la Revista:||JOURNAL OF BIOLOGICAL CHEMISTRY|
|Fecha de publicación:||2004|
|Página de inicio:||21689|