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

Abstract

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca(2+) 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 Ca(2+) channel beta subunits (Ca(v)beta), 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 Ca(2+) 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(v)beta but not the Gem/Ca(v)beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca(v)alpha(1)) in a Ca(v)beta-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(v)alpha(1) critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca(v)alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca(v)beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca(v)alpha(1) that binds Ca(v)beta). Our results challenge the prevailing view regarding the role of Ca(v)beta in RGK inhibition of high voltage-activated Ca(2+) channels and prompt a paradigm in which Gem directly binds and inhibits Ca(v)beta-primed Ca(v)alpha(1) on the plasma membrane.

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.

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.

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).

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.

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.