Direct inhibition of P/Q-type voltage-gated Ca²⁺ channels by Gem does not require a direct Gem/Ca_vβ interaction

Abstract

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca²⁺ channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca²⁺ channel β subunits (Ca_vβ), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca²⁺ channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca_vβ but not the Gem/Ca_vβ interaction, and that Gem coimmunoprecipitates with the P/Q channel α₁ subunit (Ca_vα₁) in a Ca_vβ-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca_vα₁ critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca_vα₁ abolishes Gem inhibition of P/Q channels and confers Ca_vβ-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca_vα₁ that binds Ca_vβ). Our results challenge the prevailing view regarding the role of Ca_vβ in RGK inhibition of high voltage-activated Ca²⁺ channels and prompt a paradigm in which Gem directly binds and inhibits Ca_vβ-primed Ca_vα₁ on the plasma membrane.

High voltage-activated (HVA) Ca²⁺ channels, which include L-, N-, P/Q-, and R-type channels, are essential for diverse biological processes, ranging from gene transcription and neurotransmission to hormone secretion and heart beat. They contain a pore-forming α₁ subunit (Ca_vα₁), a membrane anchored α₂δ subunit, and a cytosolic β subunit (Ca_vβ; review in ref. 1). Ca_vα₁ has four homologous repeats, each consisting of six transmembrane segments (S1–S6) and a pore-forming loop. Ca_vα₁ is the principal subunit of HVA Ca²⁺ channels and is the main determinant of the unique pharmacological and biophysical properties of each channel type. Ca_vβ is an auxiliary subunit that is indispensible for transporting Ca_vα₁ to the plasma membrane and fine-tuning channel gating (reviews in refs. 2 and 3). Both effects depend critically on the binding of Ca_vβ to the α interacting domain (AID) in the cytoplasmic loop (referred to as the I–II loop) connecting the first two homologous repeats of Ca_vα₁ (2–11). Gating regulation by Ca_vβ also needs a continuous α-helix between the AID and the S6 segment of the first repeat (IS6) of Ca_vα₁ (3, 8, 9, 12).

The activity of HVA Ca²⁺ channels is regulated by numerous signaling pathways and interacting proteins with profound functional consequences (review in ref. 1). Recently, members of the Rem, Rem2, Rad, and Gem/Kir (RGK) family of Ras-related monomeric small GTP-binding proteins, which are known to regulate cytoskeleton remodeling through the Rho/Rho kinase signaling cascade (review in ref. 13), have emerged as the most potent protein inhibitors of HVA Ca²⁺ channels (14–31). RGK proteins are present in many tissues and cells where HVA Ca²⁺ channels are expressed, including the brain and cardiac, skeletal, and smooth muscles (review in ref. 13). Accordingly, they are emerging as strong regulators of hormone secretion and cardiac and brain physiology, both in vivo and in vitro. For example, in the heart, dominant negative suppression of endogenous Rad increases L-type Ca²⁺-channel currents and action-potential duration in cardiac cells and produces longer QT intervals and arrhythmias (24). Rem2 prevents glucose-stimulated insulin secretion in pancreatic β cells (19) and regulates the development of both excitatory and inhibitory synapses, presumably through a feedback loop that controls Ca²⁺ influx (32). Finally, alteration in Gem regulation of Ca_v1.2 L-type Ca²⁺ channels may contribute to certain neural phenotypes displayed in Timothy Syndrome, a genetic disorder characterized by cardiac and neurological defects and autism (33).

All RGK proteins associate directly with Ca_vβ in vitro and in cells (14–17, 19–21, 23, 25, 27–29, 34), and Ca_vβ is required for RGK-induced inhibition of HVA Ca²⁺ channels (14, 15, 22). Two modes of action have been reported: (i) RGK proteins reduce the number of HVA Ca²⁺ channels on the cell surface, either by interfering with channel trafficking to the plasma membrane or increasing endocytosis of surface channels (14, 16, 17, 20, 24, 31, 34, 35), and (ii) RGK proteins inhibit channels already on the plasma membrane (18, 19, 21, 25, 31). The molecular mechanisms of either mode of action are unknown, but because of the central role of Ca_vβ in HVA Ca²⁺-channel trafficking and gating, it is widely assumed that both forms of inhibition rely on the RGK/Ca_vβ interaction (13–29, 31). This key hypothesis, however, has not been tested.

In this study, we investigated the molecular mechanism of Gem inhibition of P/Q-type Ca²⁺ channels expressed in Xenopus oocytes. We unambiguously show that Gem directly inhibits P/Q-type Ca²⁺ channels on the plasma membrane and that this inhibition requires Ca_vβ. Surprisingly, we discover that Gem inhibition does not require a direct Gem/Ca_vβ interaction or a structural element critical for Ca_vβ regulation of channel gating. Instead, Ca_vβ seems required only to prime Ca_vα₁ for Gem inhibition. We find that Gem and P/Q channel Ca_vα₁ coimmunoprecipitate, and we identify a region in Ca_vα₁ crucial for Gem inhibition. Our results show an essential and hitherto unrecognized contribution from Ca_vα₁ to RGK inhibition and lead us to propose a model for RGK inhibition of HVA Ca²⁺ channels.

Results

Gem Directly Inhibits P/Q-Type Ca²⁺ Channels on the Plasma Membrane.

To date, there is no report of direct inhibition of surface HVA Ca²⁺ channels by Gem. Thus, we first investigated whether Gem inhibited P/Q-type Ca²⁺ channels on the plasma membrane. To accomplish this, we designed a Gem construct (S68-K276 of human Gem) that contained most of the Gem protein and could be readily purified. Gem(S68-K276), when purified and applied to the intracellular face of giant inside-out membrane patches from Xenopus oocytes expressing P/Q channels, markedly and quickly (in ~1 min) reduced the macroscopic currents (Fig. 1). This inhibition was only partially reversible (Fig. 1 A and B), partly because of the difficulty of washing out the applied protein in this recording configuration, and it was largely voltage-independent, as indicated by the current–voltage relation (Fig. 1C Right). This result shows that Gem(S68-K276) acutely inhibits P/Q channels on the plasma membrane. As expected, Gem(S68-K276) inhibited whole-oocyte P/Q-channel currents when tonically expressed (Fig. S1).

Fig. 1.

Gem acutely inhibits surface P/Q-type Ca²⁺ channels. (A) Time course of current inhibition (recorded at +20 mV) by 5 μM purified Gem(S68-K276) in inside-out membrane patches from oocytes expressing Ca_v2.1, α₂δ, and β₃. In this figure and subsequent similar figures, currents were normalized and then averaged (n = 5). (B) Exemplar time course of inhibition in a single patch, with the same conditions as in A. (C) Current traces recorded at +20 mV (Left) and current–voltage relationships (Right) taken at the times indicated by I, II, and III in B.

Ca_vβ Is Required for Gem Inhibition of Surface P/Q-Type Ca²⁺ Channels.

We next investigated whether Ca_vβ was required for the acute and direct inhibition of surface P/Q channels by Gem. To obtain large populations of surface P/Q channels that did not contain a Ca_vβ (β-less channels), we created, following our previous strategy (12), a mutant β₃ named β₃_Mut2 (bearing the M196A/L200A mutation). β₃_Mut2 retained the ability to chaperone Ca_v2.1 to the plasma membrane but with vigorous perfusion, could quickly dissociate from the surface channels in inside-out patch recordings, leaving functional β-less channels behind. This dissociation was ascertained by monitoring the positive shift of the activation curve and its subsequent restoration (i.e., negative shift) after the application of purified β₃_core (amino acids G16–G366 of β₃) (Fig. 2A). Purified Gem(S68-K276) did not inhibit the β-less channels but quickly and strongly suppressed the same population of channels after they bound β₃_core (Fig. 2 B and C). The inhibition developed well beyond the current level before the application of β₃_core, indicating that Gem(S68-K276) did not simply reverse the gating modulation by β₃_core. These results indicate that Ca_vβ is necessary for Gem-induced acute inhibition.

Fig. 2.

Ca_vβ is required for Gem inhibition of surface P/Q-type Ca²⁺ channels. (A) Voltage-dependence of activation under the indicated conditions for channels composed of Ca_v2.1, α₂δ, and β₃_Mut2. Activation curves were obtained in inside-out patches before the wash out of β₃_Mut2, after 5 min of wash, and 1 min after subsequent application of β₃_core (n = 5). (B) Time course of Gem action on β-less and β-containing channels. Currents (recorded at +20 mV) were obtained from an inside-out patch from an oocyte expressing Ca_v2.1, α₂δ, and β₃_Mut2. Before time 0, the patch had been washed for 5 min such that the channels had lost β₃_Mut2 and become β-less; 5 μM Gem(S68-K276) had no effect on the β-less channels. Subsequent application of 4 μM purified β₃_core increased the current, which was suppressed, with partial reversibility, by the second application of 5 μM Gem(S68-K276). (C) Same plot as in B for data pooled from five patches. For each patch, the current was normalized by that at time 0.

Role of Ca_vβ in Gem Inhibition.

Why is Ca_vβ necessary for Gem inhibition of P/Q channels? Ca_vβ regulates Ca²⁺-channel gating through high-affinity binding of its guanylate kinase (GK) domain to the AID in the I–II loop of Ca_vα₁ and through low-affinity interactions involving other regions of Ca_vβ and Ca_vα₁ (2–11). Thus, it is possible that Ca_vβ’s mandatory role arises from Ca_vβ-induced gating changes and the underlying low-affinity Ca_vβ/Ca_vα₁ interactions. To test this possibility, we examined the effect of Gem on channels formed by α₂δ, β₃, and a mutant Ca_v2.1 named Ca_v2.1_7G, in which a seven-glycine linker was inserted between the AID and IS6. The hepta-glycine linker disrupts the rigid α-helix between IS6 and the AID and renders Ca_vβ completely impotent in regulating channel gating without compromising its chaperone effect (8–10, 12). However, Ca_v2.1_7G channels were inhibited by Gem(S68-K276) in both whole oocytes (Fig. 3A) and inside-out patches (Fig. 3B). Thus, Ca_vβ-induced gating changes are not a prerequisite for Gem inhibition. Furthermore, in agreement with a recent study in whole oocytes (29), we found that the GK domain of β₃ (β₃_GK) alone was sufficient to support Gem inhibition of P/Q channels in inside-out patches (Fig. 3C), indicating that low-affinity interactions engaging Ca_v2.1 and other regions of β₃ are not required.

Fig. 3.

The role of Ca_vβ in Gem inhibition of P/Q channels. (A) Inhibition of P/Q channels composed of Ca_v2.1_7G, α₂δ,and β₃ by constitutively expressed Gem(S68-K276) in whole oocytes. (B) Time course of inhibition of Ca_v2.1_7G channels by 5 μM purified Gem(S68-K276) in inside-out patches (n = 5). (C) Time course of inhibition of P/Q channels containing β₃_GK by 5 μM purified Gem(S68-K276) in inside-out patches (n = 5). (D) Western blot showing the abolishment of Gem/β₃ interaction by targeted mutations. Immunoprecipitation (IP) of Gem was carried out using an anti-HA antibody from the lysates of HEK 293T cells expressing the constructs indicated on the top of each lane. HA-Gem and Myc-β₃ coimmunoprecipitated (lane 2), but HA-Gem_Mut3 and Myc-β₃_Mut3 did not (lane 4). Similar results were obtained in two other experiments. (E) Inhibition of P/Q channels containing β₃_Mut3 by constitutively expressed WT Gem or Gem_Mut3 in whole oocytes. In this figure and other similar figures, asterisks indicate P < 0.01, the number of recordings is indicated above the bar, all recordings were obtained from the same batch of oocytes, and similar results were obtained from at least two different batches of oocytes. (F) Time course of inhibition of β₃_Mut3-containing P/Q channels by 5 μM purified Gem(S68-K276)_Mut3 in inside-out patches (n = 5).

All members of RGK proteins interact directly with Ca_vβ (14–17, 19–21, 23, 25, 27–29, 34). Thus, a prevalent hypothesis is that RGK proteins need to bind Ca_vβ to exert their inhibitory effect (13–29, 31). To test this hypothesis, we abolished Gem/β₃ interaction by simultaneously mutating to alanine three residues on Gem and three residues on β₃ that have been shown to be critical for this interaction (34). Coimmunoprecipitation confirmed that the mutant Gem (R196A/V223A/H225A named Gem_Mut3) and the mutant β₃ (D194A/D270A/D272A named β₃_Mut3) did not interact with each other (Fig. 3D and Fig. S2). This result is in agreement with a previous biochemical study showing that mutating any one of these residues abolished or severely weakened Gem/β₃ interaction (34). Strikingly, Gem_Mut3 and Gem(S68-K276)_Mut3 were fully capable of inhibiting channels formed by Ca_v2.1, α₂δ and β₃_Mut3 when either constitutively expressed (Fig. 3E) or acutely applied (Fig. 3F). These results indicate that the Gem/β₃ interaction is not required for Gem-induced acute inhibition.

Gem and Ca_vα₁ Coimmunoprecipitate.

Because Gem_Mut3 did not bind β₃_Mut3, the observation that Gem_Mut3 inhibited surface P/Q channels containing β₃_Mut3 (Fig. 3 E and F) suggested that Gem_Mut3 might directly interact with Ca_v2.1. In an initial test for this possibility, we found that Ca_v2.1 and β₃ coimmunoprecipitated with Gem (lane 3 of Fig. 4A and Fig. S3). This result is in accord with previous results showing the existence of RGK/Ca_vβ/Ca_vα₁ tripartite complexes (21, 25, 27, 34). However, coimmunoprecipitation of Ca_v2.1, β₃, and Gem could simply be a result of the Gem/β₃ and β₃/Ca_v2.1 interactions. Remarkably, however, further experiments show that Ca_v2.1 and β₃_Mut3 coimmunoprecipitated with Gem_Mut3 (Fig. 4A, lane 6), although Gem_Mut3 and β₃_Mut3 did not bind each other, as shown in Fig. 3D. Moreover, Ca_v2.1 coimmunoprecipitated with either Wild Type (WT) Gem or Gem_Mut3 in the absence of an exogenously expressed Ca_vβ (Fig. 4A, lanes 2 and 5). These results suggest that either Gem directly associates with Ca_v2.1 or Gem and Ca_v2.1 both associate with an unknown protein in the same complex. In either case, this association is Ca_vβ-independent.

Fig. 4.

Model of Gem inhibition of surface P/Q channels. (A) Gem coimmunoprecipitates with Ca_v2.1 in a Ca_vβ-independent manner. IP of Gem was carried out using an anti-HA antibody from the lysates of HEK 293T cells expressing the constructs indicated on the top of each lane. Ca_v2.1 and β₃ were detected by an anti-FLAG and anti-Myc antibody, respectively. Western blot shows coimmunoprecipitation of Gem and Ca_v2.1 (lane 2), Gem_Mut3 and Ca_v2.1 (lane 5), and Gem_Mut3, Ca_v2.1, and β₃_Mut3 (lane 6). In groups without Gem, no coimmunoprecipitation of Ca_v2.1 was observed (lanes 1 and 4). As a positive control, Ca_v2.1 and WT β₃ are coimmunoprecipitated by WT Gem (lane 3). Similar results were observed in three other experiments. (B) Model of Gem inhibition of surface P/Q channels. Gem associates directly with Ca_v2.1 through an anchoring site on Ca_v2.1 (indicated by the orange patch), with (I and III) or without (II) Ca_vβ. Binding of WT Ca_vβ (I) or β₃_Mut3 (III) to Ca_v2.1 induces an inhibitory binding site in Ca_v2.1 (indicated by the pink patch) where WT Gem (I) or Gem_Mut3 (II) binds to cause inhibition.

Model for Gem Inhibition of Surface P/Q-Type Ca²⁺ Channels.

The preceding results lead us to propose a Ca_vβ-priming model for Gem inhibition of P/Q-type Ca²⁺ channels on the plasma membrane (Fig. 4B). The most distinct feature of this model is that the interaction between Gem and Ca_vβ is not necessary for Gem's inhibitory effect, but a direct association between Gem and Ca_v2.1 is essential. In this model, Gem interacts directly with Ca_v2.1 through an anchoring site, with or without Ca_vβ present. In the presence of Ca_vβ and Gem, Ca_v2.1 forms a multimeric complex with both proteins on the plasma membrane (Fig. 4B, row I). Binding of Ca_vβ to Ca_v2.1 produces a conformational change, resulting in the formation of an inhibitory binding site in Ca_v2.1 where Gem contacts to produce inhibition (Fig. 4B, row I). When Ca_vβ dissociates or is washed off from the surface Ca_v2.1, the inhibitory binding site disappears, and Gem becomes unable to inhibit Ca_v2.1, although it can still be attached to Ca_v2.1 through the anchoring site (Fig. 4B, row II). With mutant forms of Ca_vβ and Gem that cannot bind each other (Fig. 4B, row III), inhibition can still proceed, because the ability of Ca_vβ and Gem to bind Ca_v2.1 is uncompromised. According to this model, the essential role of Ca_vβ is to convert Ca_v2.1 into a state permissive for Gem inhibition.

Identification of a Ca_vα₁ Region Critical for Gem Inhibition.

To further test the Ca_vβ-priming model, we attempted to identify the molecular determinants in Ca_vα₁ that are critical for Gem inhibition. To this end, we took advantage of the observation that RGK proteins do not regulate the activity of low voltage-activated T-type Ca²⁺ channels, which do not associate with Ca_vβ or require Ca_vβ for their activity (15, 18), and we constructed chimeras between Ca_v2.1 and Ca_v3.1, a T-type channel α₁ subunit.

Expression of Ca_v3.1 in Xenopus oocytes produced typical, small, and fast inactivating T-type currents, which were unaffected by the coexpression of β₃ (Fig. 5 A and B). Consistent with previous reports (15, 18), Gem did not affect T-type currents, either with or without the coexpression of β₃ (Fig. 5B). Replacing the I–II loop of Ca_v3.1 (from I394 to I739) with that of Ca_v2.1 (from L359 to M483) produced a mutant named T_{(PQ I-II loop)} that had larger currents (Fig. 5 C and E). Coexpression of β₃ with T_{(PQ I-II loop)} did not significantly increase the peak current amplitude but markedly slowed the speed of inactivation (Fig. 5 C and D), indicating that β₃ was able to bind T_{(PQ I-II loop)}. This result was expected, because the I–II loop of Ca_v2.1 contains the Ca_vβ-binding AID. However, coexpression of Gem with T_{(PQ I-II loop)} had no effect on the amplitude of the current, either in the presence or absence of β₃ (Fig. 5E). These results indicate that binding of β₃ to the α₁ subunit alone is not sufficient to confer Gem inhibition to T-type channels.

Fig. 5.

The region encompassing IIS1–IIS3 of Ca_v2.1 is essential for Gem inhibition. (A) Representative family of currents from WT T-type channels. (B) Comparison of peak currents in oocytes expressing Ca_v3.1 and the indicated proteins. (C) Representative family of currents from channels formed by T_{(PQ I-II loop)} (schematized in Top), with or without coexpression of β₃. (D) Comparison of the time constant of inactivation of channels formed by T_{(PQ I-II loop)} alone and by T_{(PQ I-II loop)} + β₃ (n = 5–6). (E) Comparison of peak currents in oocytes expressing T_{(PQ I-II loop)} and the indicated proteins. (F) Exemplar family of currents from channels formed by T_{(PQ I-II loop—IIS3)} (schematized in Top), with or without coexpression of β₃. (G) Comparison of the time constant of inactivation of channels formed by T_{(PQ I-II loop—IIS3)} alone and T_{(PQ I-II loop—IIS3)} + β₃ (n = 5–6). (H) Comparison of peak currents in oocytes expressing T_{(PQ I-II loop—IIS3)} and the indicated proteins, showing inhibition of channels containing T_{(PQ I-II loop—IIS3)} and β₃ by WT Gem and inhibition of channels containing T_{(PQ I-II loop—IIS3)} and β₃_Mut3 by Gem_Mut3. (I) Comparison of the effect of Gem on currents produced by WT P/Q channels formed by Ca_v2.1, α₂δ, and β₃ and mutant channels formed by PQ_{(T IIS1—IIS3)} (schematized in Upper), α₂δ, and β₃, showing the complete lack of inhibition of the mutant channels. Similar results were observed in at least three batches of oocytes.

Next, we created a chimeric Ca_v3.1 in which the I–II loop and the immediately adjacent downstream S1, S2, and S3 transmembrane segments of the second homologous repeat (from I394 to G828) were replaced by those of Ca_v2.1 (from L359 to F577); this mutant is named T_{(PQ I-II loop—IIS3)} (Fig. 5F). In the absence of an exogenous Ca_vβ, the current produced by T_{(PQ I-II loop—IIS3)} was small and unaffected by Gem (Fig. 5 F and H). Coexpression of β₃ with T_{(PQ I-II loop—IIS3)} greatly increased the peak current (Fig. 5 F and H) and slowed the inactivation speed (Fig. 5 F and G). Remarkably, in the presence of β₃, coexpression of Gem drastically suppressed the peak current (Fig. 5H). The magnitude of this inhibition is nearly as large as Gem inhibition of WT P/Q channels (Fig. 5I). Thus, by grafting the I–II loop, IIS1, IIS2, and IIS3 of Ca_v2.1 to Ca_v3.1, we were able to confer full-fledged Gem inhibition to an otherwise completely resistant T-type channel. These results indicate that IIS1, IIS2, and IIS3 are critical for Gem inhibition. They also reiterate the critical role of Ca_vβ in Gem inhibition.

We then examined whether the Gem/Ca_vβ interaction is necessary for Gem inhibition of T_{(PQ I-II loop—IIS3)} by testing the effect of Gem_Mut3. As with P/Q channels (Fig. 3 E and F), Gem_Mut3 strongly suppressed the channels formed by T_{(PQ I-II loop—IIS3)} and β₃_Mut3 (Fig. 5H), indicating that a direct physical interaction between Gem and Ca_vβ is not required for Gem inhibition.

In a complimentary experiment, we replaced a region harboring IIS1, IIS2, and IIS3 of Ca_v2.1 (from R482 to F577) with its counterpart from Ca_v3.1 (from K738 to G828); this mutant is referred to as PQ_{(T IIS1—IIS3)} (Fig. 5I). Expression of this mutant Ca_v2.1 alone in oocytes produced little or no current, much like WT P/Q channels (Fig. S4). Coexpression of β₃ greatly increased the current (Fig. S4), indicating that PQ_{(T IIS1—IIS3)} binds β₃. Strikingly, Gem had little or no effect on this current while strongly suppressing WT P/Q channel currents in the same batch of oocytes (Fig. 5I). These results identify a region in Ca_vα₁ (IIS1–IIS3) that is essential for Gem inhibition.

Discussion

In this study, we show the following key features of Gem inhibition of P/Q-type Ca²⁺ channels: (i) Gem directly inhibits channels on the plasma membrane (Fig. 1), (ii) this inhibition requires Ca_vβ (Fig. 2), (iii) this inhibition does not require a direct physical interaction between Gem and Ca_vβ or Ca_vβ-induced gating changes (Fig. 3), (iv) Gem coimmunoprecipitates with Ca_v2.1 (Fig. 4A), and (v) the region encompassing IIS1–IIS3 in Ca_v2.1 is essential for Gem inhibition (Fig. 5 H and I). Several of these key features (including ii, iii, and v) are reinforced by the results obtained from various Ca_v2.1/Ca_v3.1 chimeras (Fig. 5). These results, in aggregate, strongly support the Ca_vβ-priming model proposed in Fig. 4B.

In the first study of RGK inhibition of HVA Ca²⁺ channels, Gem was reported to suppress the activity of L-, N-, and P/Q-type channels expressed in Xenopus oocytes and baby hamster kidney cells by decreasing their expression at the cell surface (14). By directly applying purified Gem proteins to the cytoplasmic face of inside-out membrane patches containing expressed P/Q channels, we show that the surface channels are rapidly and directly inhibited by Gem. Inhibition of surface HVA Ca²⁺ channels has also been observed for two other RGK proteins. Thus, Rem2 inhibits endogenous surface N-type channels in sympathetic and dorsal-root ganglion neurons (18), Rem inhibits surface L-type channels expressed in pancreatic β-cells (19, 21) and HEK 293 cells (31), and rapid translocation of a recombinant Rem derivative acutely inhibits L- and N-type channels expressed in tsA201 cells (25).

Previous studies show that RGK proteins do not affect Ca²⁺ channels in heterologous systems expressing only Ca_vα₁ (14, 15, 22), suggesting that Ca_vβ is necessary for RGK inhibition of Ca²⁺ channels. Consistent with this notion, it has been shown and is reproduced in this study that T-type Ca²⁺ channels, which do not contain an associated Ca_vβ, are not modulated by RGK proteins (Fig. 5A) (15, 18). However, until now, it was unclear whether Ca_vβ is required for RGK inhibition of surface Ca²⁺ channels. By successfully producing large populations of β-less Ca²⁺ channels on the plasma membrane, we show unequivocally that Ca_vβ is essential for this direct inhibitory effect (Fig. 2).

A striking finding of this work is that the direct association between Gem and Ca_vβ is not necessary for Gem inhibition (Fig. 3). This finding contradicts the general belief that RGK proteins exert their inhibitory action on HVA Ca²⁺ channels through Ca_vβ (13–29, 31). However, a direct physical interaction between RGK proteins and Ca_vβ has been well-documented (14–17, 19–21, 23, 25, 27–29, 34). Therefore, what is the functional importance of this interaction? One possibility is that in native cells, with physiological levels of RGK proteins, the RGK/Ca_vβ interaction serves to facilitate RGK inhibition by bringing RGK proteins close to surface Ca²⁺ channels, hence increasing the effective local concentration of RGK proteins near the channels. Another completely disparate function of this interaction could be to translocate full-length Ca_vβ into the nucleus (20), where Ca_vβ could engage in transcriptional regulation (36).

The Ca_vβ-priming model proposed here for Gem may also be applicable to Rem and Rem2, because they both have been shown to inhibit surface Ca²⁺ channels (18, 19, 21, 25, 31). This model remains speculative, and many questions remain to be answered, including the locations of the proposed anchoring site and inhibitory binding site for Gem in Ca_vα₁. These binding sites are likely located in the cytoplasmic regions of Ca_vα₁, given that Gem is a cytosolic protein. Indeed, a new study reports that a proximal C-terminal region of Ca_v1.2 can directly bind Rem, Rem2, and Rad in vitro (30). Because this binding occurs in the absence of a Ca_vβ, it could serve as the anchoring interaction proposed in our model. This hypothesis needs further testing. A mutant Ca_v1.2 containing the S1928A mutation was reported to be resistant to inhibition by Rem2 (37), which would suggest that this C-terminal region and protein kinase A (PKA) phosphorylation of Ca_vα₁ regulate Rem2 inhibition of L-type channels. However, deleting the distal C terminus of Ca_v1.2 (from K1906 to L2171) has no effect on Rem inhibition of Ca_v1.2, although this is a region that also binds Rem in vitro (30). Thus, the role of Ca_vα₁ C terminus in RGK inhibition remains largely unclear. The molecular mechanism of the involvement of the IIS1–IIS3 region, which is highly conserved among the Ca_vα₁ of HVA Ca²⁺ channels (Fig. S5), in Gem inhibition is also unclear. One possible role of this region is mediating the conformational changes of Ca_vα₁ that likely occur after Ca_vβ and/or Gem binding.

Although many of the molecular details remain to be elucidated, our findings and the Ca_vβ-priming model shift the focus from the RGK/Ca_vβ interaction to RGK/Ca_vα₁ interactions. This shift may prompt new investigations into and expand our understanding of the molecular mechanisms of RGK regulation of HVA Ca²⁺ channels.

Materials and Methods

WT and mutant P/Q- and T-type Ca²⁺ channels were expressed in Xenopus oocytes with or without WT or mutant Gem. Ba²⁺ currents were recorded with inside-out patch clamp or two-electrode voltage clamp. Purified Gem protein fragments were applied to membrane patches in inside-out patch-clamp recordings. Coimmunoprecipitation was carried out in HEK 293T cells.

Details for the methods described above and construct cloning, cell culture and transfection, SDS/PAGE and Western blot, oocyte preparation and injection, protein expression and purification, coimmunoprecipitation, and electrophysiology are provided in SI Materials and Methods.