The Ca²⁺ channel subunit Ca_Vβ2a‐subunit down‐regulates voltage‐activated ion current densities by disrupting actin‐dependent traffic in chromaffin cells

Abstract

β‐Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore‐forming α1 subunit. However, they comprise protein–protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca2+ channels (CaVβ2a) and its SH3 domain (β2a‐SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaVβ2a or β2a‐SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole‐cell patch clamp recordings, Ca2+ currents were reduced by CaVβ2a in the presence of exogenous α1‐interaction domain. β2a‐SH3, but not its dimerization‐deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following β2a‐SH3 injection. Furthermore, β2a‐SH3 was still able to reduce Ca2+ currents when dynamin‐2 function was disrupted, but not when SNARE‐dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaVβ2a and β2a‐SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a‐SH3 emerges as a signaling module that might down‐regulate forward trafficking of ion channels by modulating actin dynamics.

Dynamin-2 Mutations Linked to Neonatal-onset Centronuclear Myopathy impair exocytosis and endocytosis in adrenal chromaffin cells

“…Bovine adrenal glands were acquired from local slaughterhouses (Frigorífico Cocarsa- Ecocarnes S.A., San Fernando, Provincia de Buenos Aires, Argentina, and Frigorífico Don Pedro, Quilpué, Chile). The isolation and culture of chromaffin cells was performed as previously published (Guerra et al, 2019). In brief, after adrenal medulla digestion with a solution containing 0.25% collagenase B, 0.01% trypsin inhibitor and 0.5% bovine serum albumin, chromaffin cells were isolated by Percoll gradient centrifugation and resuspended in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/Nutrient Ham’s Mixture supplemented with 10% fetal bovine serum.…”

The interplay between α7 nicotinic acetylcholine receptors, pannexin‐1 channels and P2X7 receptors elicit exocytosis in chromaffin cells

“…Chromaffin cells from bovine origin were isolated from adrenal glands acquired from the slaughterhouse “Frigorífico Don Pedro”, as previously described (Guerra et al., 2019). In brief, adrenal glands were digested in a solution containing 0.25% collagenase B, 0.01% trypsin inhibitor and 0.5% bovine serum albumin.…”

“…Researchers were not blinded during the experiments. Sample sizes were not statistically pre‐determined, but estimated based on previous studies (del Barrio et al., 2011; Guerra et al., 2019; Harcha et al., 2019). Results are expressed as means ± SEM .…”

Voltage‐gated calcium channel nanodomains: molecular composition and function

“…More recently, by using bovine adrenal cells as a model, Guerra and colleagues [116], observed an up-regulation of HVA Ca 2+ currents when a dominant-negative (K44A) dynamin-2 mutant was transfected. When the cells were also injected with Ca V β-SH3 peptides, HVA Ca 2+ currents were reduced, ruling out a dynamin 2-dependent pathway.…”

“…Although the precise circumstances promoting a Ca V β switch from forward to retrograde trafficking are unknown, it could be possible that the Ca V β interactions with dynamin may have a role outside of the Ca V channel complex. Indeed, Ca V β‐SH3 can regulate the expression of sodium and potassium channels at the plasma membrane [116–119], thus supporting such a possibility.…”