Facilitation of rabbit α_1B calcium channels: involvement of endogenous Gβγ subunits

Abstract

The α1B (N‐type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gβγ subunits. Using transient expression in COS‐7 cells of α1B together with the accessory subunits α2‐δ and β2a, we have examined the role of endogenous Gβγ subunits in the tonic modulation of α1B, and compared this with modulation by exogenously expressed Gβγ subunits. Prepulse facilitation of control α1B/α2‐δ/β2a currents was always observed. This suggests the existence of tonic modulation of α1B subunits. To determine whether endogenous Gβγ is involved in the facilitation observed in control conditions, the βARK1 Gβγ‐binding domain (amino acids 495‐689) was overexpressed, in order to bind free Gβγ subunits. The extent of control prepulse‐induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPβS also reduced the control facilitation. Co‐expression of the Gβ1γ2 subunit, together with the α1B/α2‐δ/β2a calcium channel combination, resulted in a marked degree of depolarizing prepulse‐reversible inhibition of the whole‐cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid‐point of the voltage dependence of activation. Activation of endogenous Gβγ subunits by dialysis with GTPγS produced a smaller degree of prepulse‐reversible inhibition. The rate of reinhibition of α1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gβ1γ2 than for the decay of facilitation in control cells. Furthermore, βARK1 (495‐689) co‐expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gβγ in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. These findings indicate that, in this system, the voltage‐dependent facilitation of α1B that is observed under control conditions occurs as a result of endogenous free Gβγ binding to α1B.

G Protein Modulation of Voltage-Gated Calcium Channels

“…Whether these processes actually result in physical dissociation and reassociation between the G protein subunits and channel remains formally to be established. However, the finding that the rate of reblock is dependent on the concentration of activated G protein is consistent with this view (Elmslie and Jones, 1994;Stephens et al, 1998a;Zamponi and Snutch, 1998). The interpretation of these results is that the process involves binding from the pool of free G␤␥ dimers.…”

Introducing an alternative biophysical method to analyze direct G protein regulation of voltage-dependent calcium channels

“…At the whole-cell level, these modifications include: i) current inhibition (Boland and Bean, 1993;Wu and Saggau, 1997), ii) slowing of activation kinetics (Marchetti et al, 1986), iii) current facilitation following membrane depolarization with strong prepulse application (Ikeda, 1991;Scott and Dolphin, 1990), and iv) shift in the voltage-dependence of activation towards depolarized values (Bean, 1989). The current inhibition is definitively linked to G protein association onto the channel, whereas the slowing of activation kinetics as well as the current prepulse facilitation are interpreted as due to G protein dissociation (Elmslie and Jones, 1994;Stephens et al, 1998). The interpretation of the shift of the voltage-dependence of activation is possibly less clear, but it might also be linked to the process of G protein dissociation during membrane depolarization (De Waard et al, 2005).…”

G Protein Regulation of Neuronal Calcium Channels: Back to the Future

“…In addition, short highly depolarizing voltage step, usually applied about 1100 mV before the current eliciting pulse (double-pulse protocol), is sufficient to reverse, at least partially, most of the landmarks of G protein inhibition and produce a so-called prepulse facilitation (Scott and Dolphin, 1990;Ikeda, 1991;Doupnik and Pun, 1994). Current inhibition has been attributed to the direct binding of Gbg dimer to the Ca v 2 subunit (referred to as ON landmark), whereas all the other landmarks, including the slowing of current kinetics and prepulse facilitation, can be described as variable time-dependent dissociation of Gbg dimer from the channel (referred to as OFF landmarks) and consequent current recovery from inhibition (Elmslie and Jones, 1994;Stephens et al, 1998;Weiss et al, 2006). It is worth noting that ON and OFF landmarks do not represent two independent regulations, but rather the transition from Gbg-bound channels to Gbg-unbound channels, and vice versa.…”

“…Hence, it was proposed that the I-II loop could be the channel molecular determinant mediating the slowing of current kinetics under direct G protein regulation, and that current inhibition per se could require other molecular determinants (Page et al, 1997). Considering that the apparent slowing of current activation kinetics is attributed to the dissociation of Gbg dimer from the channel (OFF landmarks) (Elmslie and Jones, 1994;Stephens et al, 1998;Weiss et al, 2006), the I-II loop might be the channel molecular determinant involved in the dissociation of Gbg dimer from the Ca v 2 subunit.…”