Plasma membrane targeting is essential for Rem-mediated Ca2+ channel inhibition

Abstract

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca(2+) channels and a known interacting partner for Ca(2+) channel accessory beta subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca(V)beta association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1-265)) displays reduced in vivo binding to membrane-localized beta 2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1-265) can associate with beta 2a in vitro and beta 1b in vivo, suggesting that the C terminus does not directly participate in Ca(V)beta association. Despite demonstrated beta 1b binding, Rem-(1-265) was not capable of regulating a Ca(V)1.2-beta 1b channel complex, indicating that beta subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1-265) C terminus restored membrane localization and Ca(2+) channel regulation, suggesting that beta binding and membrane localization are independent events required for channel inhibition.

Figure 1. Deletion of the Rem C-terminus prevents plasma membrane localization

(A) Diagram showing features of the Rem C-terminus and the locations of Rem truncations. (B) TsA201 cells were transfected with plasmids expressing Rem^WT and either empty pCMVT7F2 vector, Ca_V1.2 and/or Flag-Ca_Vβ2a. 72 hours post-transfection, cells were examined by confocal microscopy. The localization of Rem^WT at the cell periphery is not significantly affected by co-expression of calcium channel components. (C) TsA201 cells were transfected with plasmids expressing Rem truncations and either empty pCMVT7F2 vector, Ca_V1.2 and/or Flag-Ca_Vβ2a, as described in Figure 1B. GFP-Rem^1-265 and GFP-Rem^1-270 show cytosolic localization, GFP-Rem^1-276 shows slight cell periphery enrichment, and GFP-Rem^1-282 displays very strong cell periphery enrichment consistent with plasma membrane localization irrespective of Ca_Vβ2a or Ca_V1.2 co-transfection. (D) Confocal images were quantified by line-scan from the cytosolic interior of the cell to the plasma membrane as described under “Experimental Procedures”. Intensity at the cell periphery was divided by the mean intensity over the total line-scan to find cell peripheral enrichment. Line-scan was performed four times for each cell examined and the results averaged. A significant difference (p<0.05) between treatments is denoted by asterisks. (E) Selection of tsA201 cells from Figures 1C and 1D. Arrows indicate patches of increased GFP-Rem^1-276 expression at the cell boundary.

Figure 2. Rem membrane localization is positively correlated to PI lipid association

3xFlag-tagged Rem truncations or empty 3xFlag vector (control) were overexpressed in tsA201 cells and cell lysates were exposed to PIP strips in an overlay assay. Association of Rem truncations with spotted lipids was observed using immunoblotting with biotinylated FLAG antibody. Although Rem^1-282 and Rem^WT display robust association with phosphorylated PI lipids, further truncation of the Rem C-terminus dramatically diminishes the interaction.

Figure 3. β2a association is not sufficient for Rem-mediated Ca²⁺ channel regulation

(A) HEK293 cells were transfected with 3xHA-Rem truncations and either empty pCMVT7F2 (FLAG) vector or Flag-Ca_Vβ2a. Co-immunoprecipitation was performed with Flag antibody and interaction with Rem examined by immunoblotting with biotinylated anti-HA antibody. (B) TsA201 cells were transfected with plasmids expressing GFP-Rem^1-265, GFP-Rem^1-270, GFP-Rem^1-276, GFP-Rem^1-282, GFP-Rem^WT, or empty pEGFP-C1 as control. Lysates were pulled down onto calmodulin-sepharose beads in the presence of 2 mM CaCl₂ or 2.5 mM EGTA, beads were boiled to release bound protein, and the ability of Rem truncations to associate with calmodulin was examined by immunoblotting with anti-Rem antibody. (C) HEK293 cells were transfected with plasmids expressing Ca_V1.2, Flag-Ca_Vβ2a, and either GFP-Rem^1-270, GFP-Rem^1-276, GFP-Rem^WT or empty pEGFP-C1 as control. Current through Ca_V1.2+Ca_Vβ2a complex was examined using the whole-cell patch clamp configuration in the presence of 30 mM Ba²⁺. (D) TsA201 cells were transfected with plasmids expressing Ca_V1.2, Flag-Ca_Vβ2a, and either GFP-Rem^1-276, GFP-Rem^WT or empty pEGFP-C1 as control. Current through Ca_V1.2+Ca_Vβ2a complex was examined using the whole-cell patch clamp configuration in the presence of 30 mM Ca²⁺. (E) Currents at 5 mV from Figure 3C. A significant difference (p<0.05) between treatments is denoted by asterisks. (F) Currents at 5 mV from Figure 3D. A significant difference (p<0.05) between treatments is denoted by asterisks.

Figure 4. Rem^1-265 can bind Ca_Vβ1b but cannot regulate channel function

(A) GST or GST-tagged Rem^1-265 protein was incubated with ³⁵S labeled Ca_Vβ2a in the presence of glutathione sepharose. Bound proteins were eluted by addition of free glutathione, resolved via SDS-PAGE, and Ca_Vβ2a association observed via autoradiography. (B) TsA201 cells were transfected with plasmids expressing Rem^WT, Rem^1-265 and either empty pCMVT7F2 vector control or Flag-Ca_Vβ1b. Co-immunoprecipitation was performed with Flag antibody and interaction with Rem proteins examined by immunoblotting with biotinylated HA antibody. Rem^1-265 and Rem^WT were both capable of binding Ca_Vβ1b. (C) TsA201 cells were transfected with plasmids expressing Ca_V1.2, Flag-Ca_Vβ1b, and either GFP-Rem^1-265 or empty pEGFP-C1 as control. Current through Ca_V1.2+Ca_Vβ1b complex examined using the whole-cell patch clamp configuration. (D) Currents at 5 mV from Figure 4C. A significant difference (p<0.05) between treatments is denoted by asterisks.

Figure 5. The isolated Rem C-terminus does not inhibit Ca²⁺ channel current

(A) TsA201 cells were co-transfected with Ca_V1.2, β2a, and either RFP or RFP-Rem^266-297, and current was examined using the whole-cell patch clamp configuration. (B) Currents at 5 mV from Figure 5A. There is no significant difference between the treatments. (C) TsA201 cells expressing either RFP-Rem^WT or RFP-Rem^266-297 were analyzed 72 h after post-transfection, by confocal microscopy.

Figure 6. Membrane-targeted Rem^1-265 inhibits I_Ca

(A) Diagram showing construction of CAAX chimeric proteins and sequences of the K-Ras4B and H-Ras C-terminal and CAAX domains. (B) TsA201 cells were transfected with plasmids expressing GFP-Rem^1-265, GFP-Rem^1-265/KRas4B-CAAX, GFP-Rem^1-265/HRas-CAAX, or GFP-Rem^WT. 72 h after transfection cells were observed by confocal microscopy. Rem^1-265 shows cytosolic localization, but fusion of either of the CAAX tags results in cell peripheral distribution stronger even than that of Rem^WT and consistent with plasma membrane localization. (C) TsA201 cells were transfected with plasmids expressing Ca_V1.2, Ca_Vβ1b, and either GFP-Rem^1-265, GFP-Rem^1-265/KRas4B-CAAX, or GFP-Rem^1-265/HRas-CAAX. Although GFP-Rem^1-265/HRas-CAAX can fully inhibit the activity of this channel complex, GFP-Rem^1-265/KRas4B-CAAX shows only partial inhibition. (D) TsA201 cells were transfected with plasmids expressing Ca_V1.2, Ca_Vβ2a, and either GFP-Rem^1-265/KRas4B-CAAX, GFP-Rem^1-265/HRas-CAAX, or GFP as a control. Although GFP-Rem^1-265/KRas4B-CAAX can fully inhibit the activity of this channel complex, GFP-Rem^1-265/HRas-CAAX shows only partial inhibition. (E) Currents at 5 mV from Figure 6C. A significant difference (p<0.05) between treatments is denoted by asterisks. (F) Currents at 5 mV from Figure 6D. A significant difference (p<0.05) between treatments is denoted by asterisks.