Direct inhibition of Ca_V2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction

Highlights

• Ca_V2.3 channels are a target of modulation by RGK GTPase.

• Gem but Rem, inhibits Ca_V2.3-mediated current independently of the Ca_Vβ subunit.

• Gem promotes a Ca_Vβ-independent, dynamin-mediated endocytosis Ca_V2.3 channels.

Abstract

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca²⁺ channel β subunit (Ca_Vβ), and the binding between Ca_Vα1/Ca_Vβ appears essential for the endocytic promotion of Ca_V1.X, Ca_V2.1, and Ca_V2.2 channels. In this study, we investigated the inhibition of Ca_V2.3 channels by RGK proteins in the absence of Ca_Vβ. To this end, Xenopus laevis oocytes expressing Ca_V2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of Ca_V2.3 channels would be the only example of a Ca_Vβ independent promotion of dynamin-dependent endocytosis.

Introduction

High-voltage-activated (HVA) calcium channels are the most important pathway for the regulated calcium flux across the plasma membrane that controls fast physiological processes such a muscle contraction, neurotransmitter release, and propagation of somatic action potentials; but also much slower processes like gene expression, spine plasticity, and hormone secretion [1,2]. This multiplicity of function relies upon the modular assembly that forms the channels and controls its expression and function. The core of this assembly is the pore-forming (Ca_Vα1), that harbor two structural and functional domains: the conductive pore domain (PD) and the voltage-sensing domain (VSD). The minimal channel HVA protein complex is composed of Ca_Vα1 and the modulatory β-subunit (Ca_Vβ) that regulates channel membrane insertion and decreases the energy required to activate the channel [3]. HVA channels are targeted by a plethora of cytoplasmic proteins that regulate their expression and function [4]. Among them, the RGK GTPase (Rad, Rem, Rem2, Gem/Kir) family has arisen as the most potent inhibitor of calcium current [5,6]. All members of RGK proteins associate directly with Ca_Vβ in vitro and cells [[7], [8], [9], [10], [11], [12], [13]], and since Ca_Vβ appears to be present in all mature HVA Ca²⁺ channels, a foregone conclusion is that all RGK potentially inhibit any HVA channels.

Two main routes have been described to inhibit HVA Ca²⁺ channels by RGK proteins: i) Reducing surface expression of mature channels and ii) Inhibiting the activity of channels already in the plasma membrane. Thus, RGK appears to control channel activity by fast (ii) and slow (i) processes in parallel. However, the molecular mechanisms underlying the different routes are still a matter of controversy. A relevant limitation is that the co-expression experiment cannot separate slow from fast inhibitory mechanisms. Isolating fast inhibition was accomplished by Fan and coworkers [14] using inside-out membrane patches expressing Ca_V2.1 channels together with a Ca_Vβ subunit mutant that can be washed out from the patch that can then be exposed purified Gem. They showed that Gem binds to Ca_V2.1-α1 in the absence of Ca_Vβ subunit, but the inhibitory effect does not appear until Ca_Vβ is re-instated. These results lead them to postulate that Ca_Vβ binding exposes a site necessary for Gem's inhibition and that this inhibitory site would be distinct from a putative anchoring site mediating direct Ca_V2.1-α1/Gem association in the absence of Ca_Vβ. Comparing the inhibitory power of different RGK proteins on Ca_V1.2 and Ca_V2.2 while ablating RGK-Ca_Vβ binding, Puckerin et al. [15] revealed specificity in the Ca_Vβ -independent inhibitory potency of different RGK proteins. For instance, Rad can inhibit CaV1.2 and Ca_V2.2 channels in a Ca_Vβ independent manner, while Rem can only reduce Ca_V1.2 mediated currents. Inhibition by Gem and Rem2, on the other hand, was only observed when association with Ca_Vβ was preserved. Fan et al. [14] results show that Gem can bind to Ca_V1.2 channel macro patches Ca_V2.1, seemingly free of Ca_Vβ, but inhibition only proceeded after re-instating Ca_Vβ. Here we took advantage of the robust expression of human Ca_V2.3 channel on Xenopus oocytes in the absence of Ca_Vβ [16] to evaluate beta-independent inhibition by acute exposure to Gem or Rem. We report here that in the absence of any auxiliary subunit, Gem, but not Rem, induces the removal of channels from the plasma membrane in a dynamin-dependent fashion.