Facilitation of rabbit alpha1B calcium channels: involvement of endogenous Gbetagamma subunits.
1. The alpha1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gbetagamma subunits. Using transient expression in COS-7 cells of alpha1B together with the accessory subunits alpha2-delta and beta2a, we have examined the role of endogenous Gbetagamma subunits in the tonic modulation of alpha1B, and compared this with modulation by exogenously expressed Gbetagamma subunits. 2. Prepulse facilitation of control alpha1B/alpha2-delta/beta2a currents was always observed. This suggests the existence of tonic modulation of alpha1B subunits. To determine whether endogenous Gbetagamma is involved in the facilitation observed in control conditions, the betaARK1 Gbetagamma-binding domain (amino acids 495-689) was overexpressed, in order to bind free Gbetagamma 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, GDPbetaS also reduced the control facilitation. 3. Co-expression of the Gbeta1gamma2 subunit, together with the alpha1B/alpha2-delta/beta2a 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 Gbetagamma subunits by dialysis with GTPgammaS produced a smaller degree of prepulse-reversible inhibition. 4. The rate of reinhibition of alpha1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gbeta1gamma2 than for the decay of facilitation in control cells. Furthermore, betaARK1 (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 Gbetagamma in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. 5. These findings indicate that, in this system, the voltage-dependent facilitation of alpha1B that is observed under control conditions occurs as a result of endogenous free Gbetagamma binding to alpha1B.
Stephens
G J
GJ
Department of Pharmacology, University College London and, Royal Free Hospital School of Medicine, London WC1E 6BT, UK.
Brice
N L
NL
Berrow
N S
NS
Dolphin
A C
AC
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004558:Electric Stimulation; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007150:Immunohistochemistry; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D011817:Rabbits; D014162:Transfection
D015220:Calcium Channels; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D019204:GTP-Binding Proteins