RGS2 blocks slow muscarinic inhibition of N-type Ca2+ channels reconstituted in a human cell line

Native N-type Ca2+ channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Gαq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N-type channels in HEK293 cells (a human embryonic kidney cell line) by coexpressing cloned α1B (CaV2.2) Ca2+ channel subunits and M1 receptors. Expressed Ca2+ currents were recorded using standard whole-cell, ruptured-patch techniques. Rapid application of carbachol produced two kinetically distinct components of Ca2+ channel inhibition. The fast component of inhibition had a time constant of < 1 s, whereas the slow component had a time constant of 5-40 s. Neither component of inhibition was reduced by pertussis toxin (PTX) or staurosporine. The fast component of inhibition was selectively blocked by the Gβγ-binding region of β-adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gβγ released from Gαq/11. The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Gαq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non-hydrolysable GTPγS. Together these results suggest that RGS2 selectively blocked slow inhibition by functioning as an effector antagonist, rather than as a GTPase-accelerating protein (GAP). These experiments demonstrate that slow muscarinic inhibition of N-type Ca2+ channels can be reconstituted in non-neuronal cells, and that RGS2 can selectively block slow muscarinic inhibition while leaving fast muscarinic inhibition intact. These results identify RGS2 as a potential physiological regulator of the slow muscarinic pathway. Muscarinic inhibition of N-type calcium (Ca2+) channels has been extensively studied in rodent superior cervical ganglion (SCG) neurons. Inhibition of N-type channels occurs through several different signalling pathways linked to one or more subtypes (M1-M5) of muscarinic receptor. The best understood pathway is fast and produces a voltage-dependent, membrane-delimited form of channel inhibition (Hille, 1994; Dolphin, 1998; Ikeda & Dunlap, 1999). This fast pathway involves signalling by Gβγ dimers (Herlitze et al. 1996; Ikeda, 1996) that are activated through M2 receptors in mouse (Shapiro et al. 1999) and through M4 receptors in rat (Bernheim et al. 1992; Fernandez-Fernandez et al. 1999). A second, less understood pathway generates fast, voltage-independent inhibition (Beech et al. 1992). Recent experiments indicate that this second fast pathway involves signalling by both Gβγ and Gα (Kammermeier et al. 2000). A third muscarinic pathway generates slow, voltage-independent inhibition of N-type Ca2+ channels (Bernheim et al. 1991; Beech et al. 1992). Slow muscarinic inhibition requires the activation of M1 receptors (Bernheim et al. 1992; Shapiro et al. 1999) and involves the production of an unknown, diffusible messenger (Hille, 1994). M-type potassium (K+) channels are also inhibited through a slow pathway linked to M1 receptors. However, it remains uncertain whether this inhibition occurs through the same slow pathway that inhibits N-type Ca2+ channels. Thus, recent experiments indicate that slow muscarinic inhibition of M-type K+ channels primarily depends on Gα11, whereas slow inhibition of N-type Ca2+ channels mainly involves Gαq (Haley et al. 2000). Because slow inhibition is sustained, slowly reversible, and cannot be relieved by repetitive depolarization, it is likely to exert a profound influence upon neuronal excitability (Hille, 1994; Ikeda & Dunlap, 1999). It is therefore important to identify factors that can regulate the slow inhibitory pathway. Regulator of G protein-signalling (RGS) proteins are a recently discovered group of molecules that interact with heterotrimeric G proteins. RGS proteins each contain a recognizable core domain that binds the switch regions of certain Gα subunits (Tesmer et al. 1997; Berman & Gilman, 1998). RGS proteins are usually assumed to function as GTPase-accelerating proteins (GAPs) and to be responsible for rapid deactivation of G protein-dependent pathways (Chen et al. 2000; De Vries et al. 2000). By accelerating GTP hydrolysis, RGS proteins speed reformation of inactive GαGβγ heterotrimers and thereby attenuate signals transmitted by both Gα and Gβγ subunits (Berman & Gilman, 1998). However, RGS proteins can also function as ‘effector antagonists’ by binding to Gα subunits and physically blocking their interactions with downstream signalling molecules (Hepler et al. 1997; Yan et al. 1997). As effector antagonists, RGS proteins block signalling by Gα but not Gβγ. The relative importance of effector antagonism vs. GAP activity has been little explored. Distinguishing between these two behaviours of RGS proteins requires a system in which signalling by both Gα and Gβγ can be monitored. N-type Ca2+ channels provide such a system because they are inhibited in distinct fashions by Gα and Gβγ subunits (Ikeda & Dunlap, 1999). Recent studies have suggested that RGS proteins play important roles in Ca2+ channel physiology (Jeong & Ikeda, 1998, 2000; Diverse-Pierluissi et al. 1999; Melliti et al. 1999, 2000; Chen & Lambert, 2000; Mark et al. 2000). The goal of the present work was to determine whether an RGS protein can regulate slow muscarinic inhibition of N-type Ca2+ channels. Toward that end, we reconstituted slow muscarinic inhibition in HEK293 cells. In this system, the identity of the receptor, the channels, and the RGS protein could be controlled. We found that RGS2 selectively blocks slow muscarinic inhibition of N-type channels, while leaving fast inhibition intact or even enhanced. These results provide new insight into RGS protein function, and identify RGS2 as a potential physiological regulator of the slow muscarinic pathway.