Regulation of Maximal Open Probability Is a Separable Function of Cavβ Subunit in L-type Ca2+ Channel, Dependent on NH2 Terminus of α1C (Cav1.2α)

Abstract

β subunits (Cavβ) increase macroscopic currents of voltage-dependent Ca2+ channels (VDCC) by increasing surface expression and modulating their gating, causing a leftward shift in conductance–voltage (G-V) curve and increasing the maximal open probability, Po,max. In L-type Cav1.2 channels, the Cavβ-induced increase in macroscopic current crucially depends on the initial segment of the cytosolic NH2 terminus (NT) of the Cav1.2α (α1C) subunit. This segment, which we term the “NT inhibitory (NTI) module,” potently inhibits long-NT (cardiac) isoform of α1C that features an initial segment of 46 amino acid residues (aa); removal of NTI module greatly increases macroscopic currents. It is not known whether an NTI module exists in the short-NT (smooth muscle/brain type) α1C isoform with a 16-aa initial segment. We addressed this question, and the molecular mechanism of NTI module action, by expressing subunits of Cav1.2 in Xenopus oocytes. NT deletions and chimeras identified aa 1–20 of the long-NT as necessary and sufficient to perform NTI module functions. Coexpression of β2b subunit reproducibly modulated function and surface expression of α1C, despite the presence of measurable amounts of an endogenous Cavβ in Xenopus oocytes. Coexpressed β2b increased surface expression of α1C approximately twofold (as demonstrated by two independent immunohistochemical methods), shifted the G-V curve by ∼14 mV, and increased Po,max 2.8–3.8-fold. Neither the surface expression of the channel without Cavβ nor β2b-induced increase in surface expression or the shift in G-V curve depended on the presence of the NTI module. In contrast, the increase in Po,max was completely absent in the short-NT isoform and in mutants of long-NT α1C lacking the NTI module. We conclude that regulation of Po,max is a discrete, separable function of Cavβ. In Cav1.2, this action of Cavβ depends on NT of α1C and is α1C isoform specific.

Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

“…Omission of the ␤-subunit significantly reduced the peak LTCC current and shifted it to more depolarized values by ϳ20 -30 mV (Fig. 1, compare B to A; see Table 1), due to lesser targeting of the ␣ 1 -subunit to the membrane (46). However, as we hypothesized, ZnT-1 did not further inhibit the remaining LTCC current, yielding 104 Ϯ 1% of the value recorded in the absence of ZnT-1 under similar conditions (Fig.…”

“…Data Analysis-The current-voltage (I-V) curve was fitted with the Boltzmann equation as previously described (46). Conductance (G) was calculated according to the equation:…”

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant β1 subunits

“…It has diverse [12], distinct [15,28], and isoform-specific [6,13] effects on membrane targeting and biophysical properties of the heteromeric channel complex. The high-resolution crystal structure of β subunits together with the Ca v pore subunit's alphainteraction domain (AID) has been solved [4,30].…”

“…Taking advantage of these features of β 1 subunits, three deletion mutants (β 1a N18, β 1a N27, and β 1a N51) were constructed from a human wild-type (WT) β 1a subunit and examined electrophysiologically. We tried to minimize important confounding factors: (1) β subunit effects on surface expression and open probability were separated by analyses of both whole-cell and single-channel currents, (2) calcium-dependent inactivation was avoided by using barium ions as charge carrier, (3) the interaction between the N termini of Ca v and β subunit [16], and the involvement of the Ca v N terminus on β subunit effects [15] was minimized by using the human cardiac Ca v 1.2 isoform, which lacks all amino acids encoded by exon 1 (see [15]). …”

Further characterization of regulation of CaV2.2 by Stargazin

“…9 They also call for caution in interpreting the imaging data to assess plasma membrane levels of expressed proteins, emphasizing the need to use more than one independent method (reviewed in ref. 16). In conclusion, the inhibitory effect of Stargazin on Gβγ-mediated modulation of Ca V 2.2 is independent from, and additional to, it's effect on channel expression and trafficking.…”