Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation

Abstract

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca(2+) channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca(2+) channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca(2+) channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of Ca(V)1.2 (PCT), including the CB/IQ domain known to contribute to Ca(2+)/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the Ca(V)1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca(2+)/CaM, and this effect is not due to Ca(2+)/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca(2+) channel inhibition and slows the kinetics of Ca(2+)-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca(2+) channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca(2+) channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca(2+) currents via modulation of Ca(2+)/CaM-mediated channel inactivation kinetics.

Figure 1. Rem interacts with the proximal and distal domains of CCT

A, Schematic of the CCT truncation mutants with the Rem interaction status indicated on the right. B, TsA201 cells were transiently co-transfected with expression vectors encoding 3xFlag-tagged Rem and either pCDNA3.1+3xHAa (empty vector), HA-CCT-FL or the indicated HA-tagged CCT truncation mutants. 48 h post-transfection, cells were harvested, and cell lysate (0.5 mg) was subjected to immunoprecipitation with anti-HA antibody as described under “Materials and Methods”. The entire bound fraction or a portion of the unbound fraction (2.5 µl) was analyzed by immunoblotting with biotin-Flag to detect Rem. C, Cell lysate (5 µg) was immunoblotted with anti-HA antibody to monitor expression of CCT-FL and the corresponding truncation mutants used in panel B. D, TsA201 cells were transiently co-transfected with vectors expressing Flag-Rem and either pCDNA3.1+3xHAa, HA-Ca_v1.2 CCT or HA-Ca_v3.2 CCT. Co-immunoprecipitation was performed using HA antibody as described in B and Rem binding examined by biotin-Flag immunoblotting. Results in each panel are representative of three independent experiments.

Figure 2. In vitro association of Rem with proximal CCT

A, ³⁵S-labeled CCT truncation mutants were prepared by in vitro transcription and translation as described under “Materials and Methods”. The ³⁵S-labeled proteins were resolved on 10% SDS-PAGE, the gels dried and exposed to film for 3 h. B, Recombinant GST or GST-Rem proteins bound to glutathione-Sepharose resin were preloaded with the indicated nucleotide, and incubated with ³⁵S-labeled CCT truncation mutants for 3h at 4°C. Bound proteins were resolved on SDS-PAGE, and the dried gel exposed to film for 16–72 h. Results are representative of four independent experiments. C, Recombinant GST, GST-Rem, or GST-Rem-(1-265) proteins were preloaded with GDP, bound to glutathione-Sepharose, and incubated with ³⁵S-labeled CCT-FL or PCT (3 h at 4°C). The resin was pelleted, washed, and the bound fractions subjected to SDS-PAGE analysis. The dried gel was exposed to film for 24 h to detect bound CCT. Results are representative of three independent experiments.

Figure 3. Rem2 and Rad associate with the proximal and distal domains of CCT, but the distal domain is not required for Ca²⁺ channel inhibition

A, TsA201 cells were transiently co-transfected with expression vectors encoding 3xFlag-tagged Rem2 and either empty pCDNA3.1+3xHAa (control), HA-tagged CCT-FL or the indicated HA-tagged CCT truncation mutants. Co-immunoprecipitation was performed with anti-HA antibody and Rem2 binding examined by immunoblotting with biotinylated FLAG antibody. Results are representative of three independent experiments. B, TsA201 cells were transiently co-transfected with expression vectors encoding Flag-Rad and either HA-CCT-FL, the indicated HA-tagged CCT truncation mutants, or empty vector. Co-immunoprecipitation analysis was performed as described in A. Results are representative of three independent experiments. C, TsA201 cells were transfected with plasmids expressing Ca_v1.2(1–1905), β_1b and either GFP-Rem or unfused GFP as control. Current was examined using the whole-cell patch clamp configuration in the presence of 30 mM Ba²⁺.

Figure 4. Plasma membrane targeting is necessary for Rem:CCT association

A, TsA201 cells were transiently co-transfected with the indicated Rem and CCT expression vectors. Co-immunoprecipitation was performed with anti-HA antibody and interaction with Rem examined by immunoblotting with biotinylated FLAG antibody. Results are representative of four independent experiments. B, TsA201 cells were transiently co-transfected with the indicated plasmids and co-immunoprecipitation performed with HA antibody as described in A. Results are representative of three independent experiments. C, TsA201 cells were transfected with the indicated plasmids. Co-immunoprecipitation was performed with Flag antibody and interaction with Rem proteins examined by immunoblotting with biotinylated anti-HA antibody. Immunoprecipitates were blotted for β_1b using biotinylated Flag antibody. Results are representative of three independent experiments. D, TsA201 cells were transfected with plasmids expressing Ca_v1.2, β_1b and either GFP-Rem-(1-265)-HCAAX, GFP-Rem-(1-265)-HSAAX, or unfused GFP as control. Current was examined using the whole-cell patch clamp configuration in the presence of 30 mM Ba²⁺. E, TsA201 cells were transiently co-transfected with GST alone, GST-Rem, or GST-Rem(266–297) and PCT. GST fusion proteins were isolated using glutathione-Sepharose resin and interaction with PCT examined by immunoblotting. Results are representative of three independent experiments.

Figure 5. Ca²⁺/CaM inhibits in vitro Rem:CCT binding

A, Recombinant GST or GST-Rem proteins bound to glutathione-Sepharose were preloaded with GDP, incubated for 3 h at 4°C with ³⁵S-labeled CCT-FL (top panel) or PCT (bottom panel) in the presence of 2 mM Ca²⁺ or 5 mM EGTA with or without CaM (2 µg). The glutathione-Sepharose beads were then pelleted and washed as described under “Materials and Methods”. Bound proteins were subjected to SDS-PAGE, and the dried gel was exposed to film for 16–72 h. Results are representative of four independent experiments. B, TsA201 cells were transiently co-transfected with expression vectors encoding HA-PCT and Flag-Rem, Flag-Rem-(1-265)-KCAAX, or Flag-Rem-(1-265)-HCAAX. The ability of PCT to interact with either Rem or the indicated Rem chimeric proteins was determined by co-immunoprecipitation analysis as described under “Materials and Methods”. Results are representative of three independent experiments. C, TsA201 cells were transfected with plasmids expressing 3xFlag-Rem, 3xFlag-Rem-(1-265), 3xFlag-Rem-(1-265)-KCAAX, 3xFlag-Rem-(1-265)-HCAAX or empty p3xFlag-CMV10 as control. Cell lysates (1 mg) were pulled down using CaM-sepharose beads in the presence of 2 mM CaCl₂, bound proteins were released with two washes with assay buffer (containing 5mM EGTA), and the ability to associate with CaM was examined by immunoblotting with anti-Flag antibody. Results are representative of three independent experiments.

Figure 6. Calmodulin expression blunts Rem-mediated Ca²⁺ channel inhibition

A, TsA201 cells were transfected with the indicated plasmids, and the current was examined in the presence of 30 mM Ba²⁺ using the whole-cell patch clamp configuration. B, TsA201 cells were transfected as in panel A, and current was examined in the presence of 30 mM Ca²⁺ using the whole-cell patch clamp configuration. C, Current density at +5 mV from A. D, Current density at +20 mV from panel B. A significant difference (p<0.01, Kruskal-Wallis test followed by a post hoc Dunn’s test) between treatments is denoted by asterisks.

Figure 7. Overexpression of Rem alters the kinetics of CDI

A, Representative normalized I_Ba traces recorded from tsA201 cells transfected with the indicated plasmids for V_test +5 mV. B, Representative normalized I_Ca traces recorded from tsA201 cells transfected with the indicated plasmids for V_test +20 mV. C, Residual fraction of currents (r₆₀₀) remaining at the end of the test pulses for V_test +5 mV in the presence of 30 mM Ba²⁺. Note that there are no significant differences between conditions. D, Overexpression of Rem slows the inactivation kinetics of CDI. During an 800 ms test pulse to a series of depolarizing potentials ranging from 0 mV to 40 mV, the inactivation time constant tau was measured as described in “Materials and Methods” and the fast inactivation component tau_fast plotted against the corresponding depolarizing potentials. The error bars represent the standard error of the mean. Analysis of the results by student’s t test revealed a significant difference between treatments denoted by single (P<0.05) or double (P<0.00005) asterisks. Statistical comparisons are between the transfection of Ca_V1.2+Flag-β_2a+GFP-Rem+pKH3-CaM (filled circles) and the transfection of Ca_V1.2+Flag-β_2a+GFP+pKH3-CaM (open squares).