Folding of Active Calcium Channel β1b -Subunit by Size-exclusion Chromatography and Its Role on Channel Function

Abstract

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 ␣ 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.Changes in the intracellular calcium concentration regulate a variety of cellular functions such as neurotransmission, muscle contraction, hormone secretion, and gene expression. High threshold voltage-activated calcium channels are the main route for calcium entry in electrically excitable cells. They are membrane protein complexes composed of at least three nonhomologous subunits, the ␣ 1 -, ␤-, and the ␣ 2 /␦-subunit. Through molecular cloning, at least 10 genes encoding mammalian ␣ 1 -subunits (␣ 1A-I and ␣ 1S ) have been identified in different cell types (1). Although the ␣ 1 -subunit encompasses all the structural elements of a functional voltage-activated calcium channel, such as the ion-conduction pathway, the voltage sensor, and drug-binding sites, the ␤-subunit seems to be essential for channel performance (2) and to be acting at two levels: (i) channel expression by interfering with the ␣ 1 -subunit endoplasmic reticulum (ER) 1 -retention signal to facilitate intracellular trafficking (3, 4), and (ii) channel activity by modifying the electrophysiological properties of the channel (5-7).Two highly conserved sequences have been identified as the primary interaction site between the ␣ 1 -and the ␤-subunit, the ␣ 1 -subunit interaction domain (AID) that lies within the c...

Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for β1 But Not for Palmitoylated β2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties

“…However, the G max was more affected than the plasma membrane expression of Ca V 2.2 (81 and 62% reduction, respectively, when coexpressed with Ca V ␤1b). The reason for this is likely to be that Ca V ␤ not only traffics the channel to the surface but also increases the maximum open probability of the channel, as suggested previously (Wakamori et al, 1999;Meir et al, 2000;Neely et al, 2004).…”

“…However, the mechanism for this increase remains controversial, being attributed to increased trafficking (Bichet et al, 2000), increased maximum open probability (Neely et al, 2004), or both. Our data agree with previous studies showing biochemically that the amount of Ca V 1.2 channels in the plasma membrane was increased by Ca V ␤ subunits (Altier et al, 2002;Cohen et al, 2005).…”

14-3-3τ Promotes Surface Expression of Cav2.2 (α1B) Ca2+ Channels

“…The ␤ subunit has been proposed to both enhance the functional expression and influence the biophysical properties of the Cav1 and Cav2 channels. Whereas some studies have described that ␤ subunit hyperpolarizes the voltage dependence of activation, augments the maximal open probability, and consequently results in increased current through individual channel and macroscopic current density (3,10,30,31), others observed that the ␤ subunit either promotes the insertion of Cav channels into the plasma membrane, as determined by gating charge measurements, imaging, and biochemical methods (7,(32)(33)(34)(35)(36)(37) or has no effect on membrane insertion at all (38). The mechanism of the Cav ␤ subunit effect on surface expression has generally been attributed to masking an ER retention signal in the ␣1 subunits (7,8).…”

Swapping the I-II intracellular linker between L-type CaV1.2 and R-type CaV2.3 high-voltage gated calcium channels exchanges activation attributes

“…Since Ca V β modulates both chimeras in a canonical fashion, our results are still consistent with the view that Ca V β increases channel activity through a coil-to-helix transition of this region. 26,40 However, the sequence content as well as the secondary structure of the I-II linker appears to be determinants of the efficiency in the coupling between voltage sensor and channel opening.…”

“…All capped cRNA were synthesized and Xenopus laevis oocytes were prepared, injected and maintained as previously reported. 40 Electrophysiological recordings were performed at 22°C with the cut-open oocyte technique four to six days after cRNA injection as described. 31 The internal recording solution contained 120 mM n-methylglucamine, 10 EGTA mM and 10 mM HEPES, pH 7.0 and the external solution contained 10 mM Ba 2+ , 96 mM n-methylglucamine and 10 mM HEPES, pH 7.0.…”