Neuronal ␣1E Ca channel subunits are widely expressed in mammalian brain, where they are thought to form R-type Ca channels. Recent studies have demonstrated that R-type channels contribute to neurosecretion and dendritic Ca influx, but little is known concerning their modulation. Here we show that ␣1E channels are strongly stimulated, and only weakly inhibited, through M1 muscarinic acetylcholine receptors. Both forms of channel modulation are mediated by pertussis toxin-insensitive G-proteins. Channel stimulation is blocked by regulator of G-protein signaling 2 (RGS2) or the C-terminal region of phospholipase C-␤1 (PLC␤1ct), which have been previously shown to function as GTPase-activating proteins for G␣q. In contrast, RGS2 and PLC␤1ct do not block inhibition of ␣1E through M1 receptors. Inhibition is prevented, however, by the C-terminal region of ␤-adrenergic receptor kinase 1, which sequesters G␤␥ dimers. Thus, stimulation of ␣1E is mediated by a pertussis toxininsensitive G␣ subunit (e.g., G␣q), whereas inhibition is mediated by G␤␥. The ability of RGS2 and PLC␤1ct to selectively block stimulation indicates these proteins functioned primarily as effector antagonists. In support of this interpretation, RGS2 prevented stimulation of ␣1E with non-hydrolyzable guanosine 5Ј-0-(3-thiotriphosphate). We also report strong muscarinic stimulation of rbE-II, a variant ␣1E Ca channel that is insensitive to voltage-dependent inhibition. Our results predict that G␣q-coupled receptors predominantly stimulate native R-type Ca channels. Receptor-mediated enhancement of R-type Ca currents may have important consequences for neurosecretion, dendritic excitability, gene expression, or other neuronal functions. Key words: Ca V 2.3; R-type calcium channel; ␣1E; RGS protein; phospholipase C-␤1; GAP; effector antagonistNative R-type Ca channels have been defined by their resistance to selective antagonists of L-, N-, and P/Q-type Ca channels (Randall and Tsien, 1995). Only recently has a selective antagonist of R-type channels been reported (Newcomb et al., 1998). Antisense depletion experiments suggest that neuronal R-type Ca channels are formed by ␣1E subunits (Piedras-Rentería and Tsien, 1998;Tottene et al., 2000). ␣1E subunits are widely expressed in mammalian brain (Niidome et al., 1992;Soong et al., 1993;Wakamori et al., 1994;Williams et al., 1994;Yokoyama et al., 1995), and several splice variants have been described (cf. Pereverzev et al., 1998). Although the physiological functions of R-type Ca channels are incompletely known, available evidence indicates that they contribute to dendritic Ca influx (Kavalali et al., 1997) and neurosecretion by some presynaptic terminals (Turner et al., 1995;Wu et al., 1998Wu et al., , 1999Allen, 1999;Wang et al., 1999).The G-protein-dependent modulation of N-and P/Q-type Ca channels has been extensively studied (for review, see Hille, 1994;Jones and Elmslie, 1997;Zamponi and Snutch, 1998;Ikeda and Dunlap, 1999;Bean, 2000). In contrast, much less is known concerning the modulation of R-t...

In rat pituitary GH3 ceils, epidermal growth factor (EGF) and insulin stimulate prolactin production, whereas glucocorticoids exert the opposite effect. In the present study, GH3 cells were subjected to whole-cell patch clamp to assess the chronic actions of such regulatory factors on voltage-dependent calcium currents. Before the electrical recording, cells were grown 5-6 d either under standard conditions or in the presence of 5 nM EGF, 100 nM insulin, 1 ~M dexamethasone or 5 I~M cortisol. EGF induced a twofold selective increase in high-threshold calcium current density. Insulin and glucocorticoids, on the other hand, specifically regulated low-threshold Ca channels. Current density through these channels increased by 70% in insulin-treated cells, and decreased by 50% in cells exposed to dexamethasone or cortisol. Other Ca channel properties investigated (conductancevoltage curves, deactivation rates, time course and voltage dependence of lowthreshold current inactivation) were unaffected by the chemical messengers. The alterations in current density persisted for many hours after removing the regulatory factors from the culture medium. In fact, the stimulatory action of EGF on high-threshold current lasted > 3 d. The results suggest that the control of prolactin production by the factors tested involves regulation of the surface density of functional Ca channels in the plasma membrane.

Regulators of G protein signaling (RGS) proteins bind to the α subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among Gα, Gβγ, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein–coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing α1B, α2, and β3 Ca channel subunits and m2 receptors, carbachol (1 μM) inhibited whole-cell currents by ∼80% compared with only ∼55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose–response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being ∼18 nM in control cells vs. ∼150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some Gα subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein–modulated Ca channels.

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.