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J. Biol. Chem., Vol. 282, Issue 39, 28431-28440, September 28, 2007

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Plasma Membrane Targeting Is Essential for Rem-mediated Ca²⁺ Channel Inhibition^*

Robert N. Correll, Chunyan Pang, Brian S. Finlin, Alexandria M. Dailey, Jonathan Satin¹, and Douglas A. Andres²

From the Departments of Molecular and Cellular Biochemistry and Physiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509

Received for publication, July 26, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The small GTPase Rem is a potent negative regulator of high voltage-activated Ca²⁺ channels and a known interacting partner for Ca²⁺ channel accessory subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca_V 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 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 2a in vitro and 1b in vivo, suggesting that the C terminus does not directly participate in Ca_V association. Despite demonstrated 1b binding, Rem-(1–265) was not capable of regulating a Ca_V1.2-1b channel complex, indicating that 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 Ca²⁺ channel regulation, suggesting that binding and membrane localization are independent events required for channel inhibition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
High voltage-activated Ca²⁺ channels (Ca_V1 and Ca_V2 families) transduce electrical activity into increased intracellular calcium that mediates a diverse array of essential cellular processes, including hormone secretion, neurotransmitter release, and excitation-contraction coupling in muscle systems (1). The cardiac L-type Ca²⁺ channel is a multiprotein complex consisting of the pore-forming Ca_V1.2 subunit and auxiliary subunits, including Ca_V and ₂- subunits (1). The Ca_V subunit determines the ion selectivity and single channel conductance of the mature channel, whereas co-expression of Ca_V or ₂ facilitates cell surface trafficking of the ₁ subunit, increases Ca²⁺ current amplitude, and alters channel gating properties (1, 2). Ca_V subunits are encoded by four genes (1–4), each subject to complex splicing (3). Ca_V2a, a isoform found in the heart, is subject to post-translational palmitoylation, which directs plasma membrane localization, whereas other isoforms are predominantly localized to the cytosol when not bound to Ca_V1 (3).

Recently, members of the RGK³ family of Ras-related GTPases, including Rem (4), Rem2 (5), Rad (6), and Gem/Kir (7), have been identified as potent regulators of HVA Ca²⁺ channel function (8–10). Although all RGK GTPases associate with Ca_V subunits and prevent de novo expression of L-type I_Ca (8–10), the mechanism of RGK protein-mediated Ca²⁺ channel inhibition remains controversial. It was originally hypothesized that RGK protein binding blocked Ca_V1/ association, leading to a reduction of functional channels at the cell surface (8, 11–14). However, a series of recent studies suggests instead that the majority of RGK proteins inhibit the activity of the preassembled channel complex at the plasma membrane (10, 15, 16), although Ca_V association still appears critical (16, 17). Moreover, RGK-mediated channel regulation appears more complex than simple Ca_V sequestration (16, 17) and may include contributions from both the Ca_V1 C terminus and protein kinase A signaling pathways (18).

The conserved RGK C terminus plays a crucial role in Ca²⁺ channel regulation. Deletion of the Rem, Rem2, and Rad C terminus inhibits plasma membrane localization of the proteins, greatly reduces Ca_V2a subunit binding, and eliminates Ca²⁺ channel regulation (9, 15, 19). Recent work has described mutations to the C-terminal domain that alter CaM and 14-3-3 binding in all RGK proteins (12–14, 20), and research by Beguin et al. (12–14) suggests that loss of CaM binding leads to nuclear localization, whereas overexpression of 14-3-3 proteins promotes the clearance of RGK proteins from the nucleus. Mutations that prevent 14-3-3 and CaM binding in Rad result in the redistribution of Rad and Ca_V3 to the nucleus (14). A corresponding loss of Rad-mediated Ca²⁺ channel regulation for these mutants has led to the suggestion that RGK-mediated channel inhibition involves nuclear targeting of Ca_V subunits (14). Thus, although it is clear that the conserved RGK C terminus plays a role in channel regulation, the exact mechanism of action remains to be determined.

Here, we analyze the contribution of the Rem C terminus to Ca²⁺ channel regulation. We find that Rem is trafficked to the plasma membrane and associates with phosphatidylinositol lipids and that truncation of the C terminus results in redistribution to the cytosol, accompanied by a loss of calmodulin binding and Ca²⁺ channel inhibition. These truncation mutants display a reduction in Ca_V2a but not Ca_V1b association in vivo, and loss of the C terminus does not affect in vitro 2a subunit binding, indicating that subunit interaction does not require the Rem C terminus. In addition, the Rem-(1–265) truncation mutant that binds Ca_V1b does not inhibit current expression from the heterologously expressed Ca_V1.2-Ca_V1b channel, indicating that Rem does not inhibit channel function solely through subunit sequestration. Anchoring of Rem-(1–265) to the plasma membrane using the CAAX motif from H-Ras or K-Ras4B restores Ca²⁺ channel inhibition, suggesting that plasma membrane localization is critical for Rem-mediated Ca²⁺ channel regulation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmids—Mammalian expression vectors for Ca_V1.2 subunit, FLAG epitope-tagged 2a subunit, FLAG epitope-tagged 1b subunit, and HA epitope-tagged Rem have been described previously (9). Rem truncation mutants were generated by PCR using HA-tagged Rem as the template and fully sequenced. RFP-Rem-(266–297) was generated by PCR and inserted behind RFP in pDsRed vector (Clontech). Chimeric Rem proteins were generated by ligation of oligonucleotides corresponding to the C terminus of human K-Ras4B (residues 171–188) or mouse H-Ras (residues 171–189) to the C terminus of pcDNA3.1 + zeo 3xHA-Rem-(1–265) utilizing XbaI/ApaI sites.

Confocal Imaging—Confocal imaging of green fluorescent protein (GFP)-tagged Rem truncations, chimeric Rem proteins, RFP-Rem-(266–297) and Rem^WT was performed as previously described (18). Images displayed are representative of the cells observed. Quantification was performed using Leica LCS software. Plasma membrane localization was quantified by four line scan intensity measurements through each cell beginning in the central cytoplasm, avoiding the nucleus, and ending at the cell periphery. GFP intensity at the cell periphery in each scan was divided by the mean intensity over the entirety of the scanned line to monitor GFP cell periphery intensity over that of the GFP-tagged protein in the cytosol. Line scans were averaged for each cell, and the mean values of the averaged cell measurements are reported as mean ± S.E. Significance was determined using Student's t test with a p value of <0.05. To examine the localization of GFP-Rem-(1–276) at the cell periphery, a double-blind study was performed. From the line scan analysis above, 32 cells expressing Rem-(1–276) and 33 cells expressing Rem-(1–265) were randomized and examined by three individuals, who were asked to score each cell for the presence of increased punctate GFP fluorescence at the cell periphery. Scored cells were then matched to their appropriate treatment, and the percentage of cells from each treatment displaying localized increases of GFP fluorescence at the cell boundary was determined. Values are reported as mean ± S.D., and significance was determined using Student's t test with a p value of <0.05.

PIP Binding Assay—3x FLAG-tagged Rem truncations or empty 3x FLAG vector was expressed in tsA201 cells using the calcium phosphate transfection method as described previously (21). 48 h post-transfection, cells were harvested and lysed in PIP binding buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 100 mM NaCl, 0.5% Triton X-100, 1x protease inhibitor mixture I (Calbiochem)), sonicated, and centrifuged at 100,000 x g. PIP strips (Molecular Probes) were blocked in TBS-T + 3% fatty acid-free bovine serum albumin for 1 h and incubated with total cell lysate from the appropriate treatment in TBS-T + 3% fatty acid-free bovine serum albumin at 4 °C overnight with gentle rocking. Membranes were then washed with TBS-T supplemented with 3% fatty acid-free bovine serum albumin and probed with biotinylated FLAG antibody and horseradish peroxidase-conjugated streptavidin. Binding of Rem truncations was detected using enhanced chemiluminescence reagent (Pierce).

Subunit Association Assays—Co-immunoprecipitation of 3x HA-tagged Rem truncations, chimeric Rem proteins, and Rem^WT with Ca_V2a and Ca_V1b in HEK293 and tsA201 cells were performed as previously described (9, 21).

Calmodulin Binding—TsA201 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and transfected with the indicated plasmids using the calcium phosphate method as previously described (21). 48 h post-transfection, cells were harvested and lysed in calmodulin immunoprecipitation buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 1x phosphatase inhibitor mixture II (Calbiochem), 1x protease inhibitor mixture I (Calbiochem), 1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 100,000 x g. Calmodulin-Sepharose beads (GE Healthcare) were washed twice with immunoprecipitation buffer and incubated with 1 mg of total lysate in the presence of 2 mM CaCl₂ or 2.5 mM EGTA. Beads were washed three times in immunoprecipitation buffer containing 2 mM CaCl₂ or 2.5 mM EGTA as appropriate, and proteins were released from the beads by boiling for 5 min in 20 µl of 2x SDS-PAGE loading buffer. Associated proteins were resolved on 10% SDS-PAGE minigels and transferred to nitrocellulose membranes. Interaction of Rem proteins with calmodulin was examined by immunoblot with Rem polyclonal antibody (9).

In Vitro Rem Binding Assay—Generation of GST-tagged Rem-(1–265) vector as well as protein production and purification have been previously described (4, 9). Generation of in vitro transcribed/translated ³⁵S-labeled Ca_V2a and the in vitro binding assay with GST-tagged Rem have been previously described (16).

Electrophysiology—HEK293 cells were transfected using Effectene (Qiagen) according to the manufacturer's instructions. TsA201 cells were transfected with the indicated plasmids using the calcium phosphate method as previously described (21), and whole-cell patch clamp experiments were performed as described previously (21). Pipette solutions consisted of 150 mM CsCl, 1 mM MgCl₂, 5 mM Mg-ATP, 3 mM EGTA, 5 mM Hepes (pH 7.36). The bath solution for Ba²⁺ recordings consisted of 112.5 mM CsCl, 30 mM BaCl₂, 1 mM MgCl₂, 10 mM tetraethylammonium chloride, 5 mM glucose, 5 mM Hepes (pH 7.4). The bath solution for Ca²⁺ recordings consisted of 112.5 mM CsCl, 30 mM CaCl₂, 1 mM MgCl₂, 10 mM tetraethylammonium chloride, 5 mM glucose, 5 mM Hepes (pH 7.4). Traces were analyzed using Origin statistical software. Values are reported as normalized mean at 5 mV ± S.E. for Ba²⁺ currents and as normalized mean at 15 mV ± S.E. for Ca²⁺ currents, and significance was determined using Student's t test with a p value of <0.05. Voltage curves were fit to the Boltzmann form.

(Eq. 1)

Electrophysiological parameters of the analyzed currents are reported in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Electrophysiological parameters of analyzed currents The Boltzmann form used was as follows: I(V) = Gmax x (V – Erev)/(1 + exp(V – V)/k).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To identify the structural domain in Rem responsible for plasma membrane trafficking, we generated a series of Rem C-terminal truncation mutants fused to GFP and examined their subcellular distribution using confocal microscopy in the presence of co-expressed empty FLAG vector control, FLAG-Ca_V2a, Ca_V1.2, or FLAG-Ca_V2a + Ca_V1.2 (Fig. 1C). Once again, co-expression of Ca_V subunits had no measurable effect on Rem mutant localization (Fig. 1C). Intensity profiling analysis (Fig. 1D) revealed that both GFP-Rem-(1–282) (5.05 ± 0.36, n = 40) and GFP-Rem^WT (2.89 ± 0.30, n = 28) were prominently localized to the cell periphery in a manner consistent with plasma membrane localization and, surprisingly, that Rem-(1–282) displayed significantly stronger targeting than Rem^WT (p < 0.001), perhaps suggesting that the distal C terminus plays a regulatory role in Rem localization. GFP-Rem-(1–276) (1.22 ± 0.02, n = 43) displayed only a slight enrichment at the cell periphery using this analysis; however, this truncation did show a statistically significant increase in membrane localization when compared with Rem-(1–270) (1.03 ± 0.01, n = 58) or Rem-(1–265) (0.99 ± 0.02, n = 55), which were expressed exclusively in the cytosol (p < 0.001) (Fig. 1, C and D). To understand this difference, we more closely examined the distribution of the GFP-Rem truncations by double-blind trial and noted that rather than a uniform membrane pattern of fluorescence, 73.96 ± 12.63% of cells expressing GFP-Rem-(1–276) displayed a punctate pattern of fluorescence at the cell boundary (Fig. 1E), as compared with 15.15 ± 13.21% of cells expressing GFP-Rem-(1–265) (p < 0.01). Taken together, these data suggest that residues 270–282 within the Rem C terminus play a critical role in targeting Rem to the plasma membrane.

View larger version (30K): [in this window] [in a new window]   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 RemWT and empty pCMVT7F2 vector, CaV1.2, and/or FLAG-CaV2a. 72 h post-transfection, cells were examined by confocal microscopy. The localization of RemWT 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 empty pCMVT7F2 vector, CaV1.2, and/or FLAG-CaV2a, as described in B. 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 CaV2a or CaV1.2 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 were averaged. A significant difference (p < 0.05) between treatments is denoted by asterisks. E, selection of tsA201 cells from C and D. The arrows indicate patches of increased GFP-Rem-(1–276) expression at the cell boundary.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Rem membrane localization is positively correlated to PI lipid association. 3x FLAG-tagged Rem truncations or empty 3x FLAG vector (control) were overexpressed in tsA201 cells and cell lysates were exposed to PIP strips in an overlay assay. Spotted lipids are identified in the above figure as follows: 1, lysophosphatidic acid; 2, lysophosphatidylcholine; 3, phosphatidylinositol (PtdIns); 4, PtdIns(3)P; 5, PtdIns(4)P; 6, PtdIns(5)P; 7, phosphatidylethanolamine; 8, phosphatidylcholine; 9, sphingosine 1-phosphate; 10, PtdIns(3,4)P2; 11, PtdIns(3,5)P2; 12, PtdIns(4,5)P2; 13, PtdIns(3,4,5)P3; 14, phosphatidic acid; 15, phosphatidylserine; 16, blank. Association of Rem truncations with spotted lipids was observed using immunoblotting with biotinylated FLAG antibody. Although Rem-(1–282) and RemWT display robust association with phosphorylated PI lipids, further truncation of the Rem C terminus dramatically diminishes the interaction.

Interestingly, although co-expression of either Rem^WT or Rem-(1–282) has been shown to result in a complete blockade of ionic current expression (9), neither Rem-(1–270) nor Rem-(1–276) was capable of generating a complete channel block in the presence of 30 mM Ba²⁺ (Fig. 3, C and E). Whole cell currents elicited in the presence of GFP-Rem-(1–270) co-expression (-11.877 ± 4.128, n = 9) were statistically indistinguishable from control currents in HEK293 cells expressing Ca_V1.2 + FLAG - _2a + GFP (-9.326 ± 1.914, n = 7) (Fig. 3E), suggesting that this truncation mutant has lost the ability to regulate Ca²⁺ channel activity. On the other hand, currents measured in the presence of Rem-(1–276) co-expression (-1.326 ± 0.627, n = 7) are 86% lower than control currents (p < 0.01) (Fig. 3E) but did not result in the complete block of current seen with Rem^WT. Since Rem-(1–276) displayed a slight but statistically significant increase in cell periphery localization when compared with Rem-(1–265) and Rem-(1–270) (Fig. 1D), it is possible that the difference in Ca²⁺ channel inhibition is due to a defect in membrane localization.

Recent studies have suggested that calmodulin association is critical for both Gem and Rad-dependent Ca²⁺ channel regulation (12, 14, 20), but the importance of calmodulin to Remmediated channel regulation is less clear (14). To explore this issue, we next examined the ability of the Rem truncations to regulate Ca_V1.2-Ca_V2a channel complexes with 30 mM Ca²⁺ as charge carrier. Although GFP-Rem-(1–276) was not capable of completely inhibiting current expression in this system (-1.212 ± 0.609, n = 13), currents obtained for Ca_V1.2 + FLAG-2a + GFP-Rem-(1–276) were not significantly different from those seen in the presence of GFP-Rem^WT (-0.493 ± 0.258, n = 9), most likely due to the smaller currents expressed in this system (Fig. 3, D and F). As seen in Fig. 3B, in a calmodulin-Sepharose binding assay, only Rem^WT and Rem-(1–282) displayed Ca²⁺-dependent calmodulin binding. Since Rem-(1–276) is capable of partial channel regulation, these data suggest that calmodulin association is not required for Rem-mediated Ca²⁺ channel regulation.

Ca_V Association Is Not Sufficient for Rem-mediated Ca²⁺ Channel Inhibition—We next investigated whether the C terminus directly contributed to Ca_V2a association or if the effect was indirect, resulting from relocalization of Rem to the cytosol. To this end, the ability of recombinant ³⁵S-labeled Ca_V2a to associate with recombinant GST-Rem-(1–265) was examined. As shown in Fig. 4A, in the absence of a cellular context, radiolabeled Ca_V2a displays binding to GST-Rem-(1–265). To extend this analysis, we next asked whether the Rem C terminus was necessary for in vivo association with a subunit isoform (Ca_V1b), which, like Ca_V2a, is localized to the plasma membrane but is not palmitoylated and is thought to be targeted to the cell surface through its C terminus (3). Lysates from tsA201 cells co-expressing HA-tagged Rem-(1–265) or Rem^WT and empty vector (control) or FLAG-tagged Ca_V1b were subjected to anti-FLAG immunoprecipitation analysis, and bound HA-tagged proteins were visualized by SDS-PAGE and immunoblotting. HA-Rem-(1–265) and HA-Rem^WT proteins bind Ca_V1b with approximately equal efficiency (Fig. 4B), demonstrating that the Rem C terminus plays no direct role in Ca_V1b binding in vivo.

To determine whether Ca_V1b binding was alone sufficient to regulate channel function, we next examined the ability of both Rem^WT and Rem-(1–265) to regulate Ca_V1.2-Ca_V1b channel current expression. Consistent with previous studies (16), tsA201 cells transiently co-transfected with GFP-tagged Rem^WT, Ca_V1.2, and Ca_V1b resulted in a complete loss of detectable ionic current expression (0.407 ± 0.392, n = 8) (Fig. 4, C and D). In contrast, currents measured from cells co-expressing channel components along with Rem-(1–265) (-10.043 ± 2.837, n = 14) were significantly different (p < 0.01) and displayed no inhibition of Ca²⁺ channel activity (Fig. 4, C and D). Taken together, these data indicate that Ca_V subunit binding alone is not sufficient for Rem-mediated Ca²⁺ channel blockade and suggest that plasma membrane localization is a critical aspect of Rem-mediated channel regulation.

The Isolated Rem C Terminus Does Not Regulate Channel Function—To determine whether the isolated Rem C terminus was sufficient for Ca²⁺ channel regulation, tsA201 cells were co-transfected with Ca_V1.2, Ca_V2a, and either empty RFP or RFP-Rem-(266–297), and currents were determined using the whole cell configuration of the patch clamp technique. Co-expression of RFP-Rem-(266–297) (-17.712 ± 5.069, n = 7) resulted in current not significantly different from that seen for channel components co-expressed with RFP (-22.275 ± 11.036, n = 6) (Fig. 5A; current at 5 mV displayed in Fig. 5B), indicating that the isolated Rem C terminus cannot regulate channel function. Confocal microscopy revealed that in contrast to full-length Rem^WT, Rem-(266–297) was found predominantly in punctate nuclear structures (Fig. 5C), suggesting that in the absence of the Rem GTP-binding core, the C terminus acts as a nuclear localization signal.

Plasma Membrane Localization Is Critical for Rem-mediated Ca²⁺ Channel Inhibition—To explore whether Rem-dependent Ca²⁺ channel regulation requires molecular contacts between the C terminus and known binding partners, such as calmodulin and 14-3-3, as suggested by recent studies (12–14), two chimeric proteins were created in which the C terminus of K-Ras4B and H-Ras were fused to Rem-(1–265) (Fig. 6A). The resulting proteins were designated Rem-(1–265)/KRas4B-CAAX and Rem-(1–265)/HRas-CAAX. The K-Ras4B C terminus is a well characterized membrane-targeting domain that contains a C-terminal polybasic domain and a farnesylation motif and displays both PI lipid and calmodulin binding, maintaining many of the functional properties of the Rem C terminus (23–25). On the other hand, the H-Ras targeting domain lacks a polybasic domain and does not bind calmodulin (24, 25). Confocal imaging of GFP-tagged versions of both Rem-(1–265)/KRas4B-CAAX and Rem-(1–265)/HRas-CAAX displayed prominent localization to the cell periphery in a manner consistent with plasma membrane localization (Fig. 6B), and both proteins were found to co-immunoprecipitate with2a (data not shown).

View larger version (40K): [in this window] [in a new window]   FIGURE 3. 2a association is not sufficient for Rem-mediated Ca2+ channel regulation. A, HEK293 cells were transfected with 3x HA-Rem truncations and either empty pCMVT7F2 (FLAG) vector or FLAG-CaV2a. Co-immunoprecipitation was performed with FLAG antibody, and interaction with Rem was 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-RemWT, 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 CaV1.2, FLAG-CaV2a, and either GFP-Rem-(1–270), GFP-Rem(1–276), GFP-RemWT, or empty pEGFP-C1 as control. Current through CaV1.2-CaV2a 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 CaV1.2, FLAG-CaV2a, and either GFP-Rem-(1–276), GFP-RemWT, or empty pEGFP-C1 as control. Current through the CaV1.2-CaV2a complex was examined using the whole cell patch clamp configuration in the presence of 30 mM Ca2+. E, currents at 5 mV from C. A significant difference (p < 0.05) between treatments is denoted by asterisks. F, currents at 5 mV from D. A significant difference (p < 0.05) between treatments is denoted by asterisks.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Rem-(1–265) can bind CaV1b but cannot regulate channel function. A, GST or GST-tagged Rem-(1–265) protein was incubated with 35S-labeled CaV2a in the presence of glutathione-Sepharose. Bound proteins were eluted by the addition of free glutathione and resolved via SDS-PAGE, and CaV2a association was observed via autoradiography. B, TsA201 cells were transfected with plasmids expressing RemWT, Rem-(1–265), and either empty pCMVT7F2 vector control or FLAG-CaV1b. Co-immunoprecipitation was performed with FLAG antibody and interaction with Rem proteins examined by immunoblotting with biotinylated HA antibody. Rem-(1–265) and RemWT were both capable of binding CaV1b. C, TsA201 cells were transfected with plasmids expressing CaV1.2, FLAG-CaV1b, and either GFP-Rem-(1–265) or empty pEGFP-C1 as control. Current through the CaV1.2-CaV1b complex was examined using the whole cell patch clamp configuration. D, currents at 5 mV from C. A significant difference (p < 0.05) between treatments is denoted by asterisks.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. The isolated Rem C terminus does not inhibit Ca2+ channel current. A, TsA201 cells were co-transfected with CaV1.2, 2a, 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 A. There is no significant difference between the treatments. C, TsA201 cells expressing either RFP-RemWT or RFP-Rem-(266–297) were analyzed 72 h after post-transfection by confocal microscopy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Since Rem directly binds to a variety of accessory Ca_V subunits (9), and a number of studies suggest that this interaction is required for the regulation of functional Ca²⁺ channels at the plasma membrane (8, 9, 11, 16), we examined whether Rem localization would be altered by co-expression of either Ca_V1.2 or Ca_V subunits or in the presence of a functional Ca_V1.2-2a Ca²⁺ channel. However, a similar fluorescence pattern was seen whether Ca_V and/or Ca_V subunits were present or absent in tsA201 cells (Fig. 1B), indicating that plasma membrane trafficking of Rem is not dependent on Ca²⁺ channel subunit expression. Beguin et al. (12–14) report that wild-type RGK proteins display cytoplasmic, plasma membrane, and prominent nuclear localization when overexpressed in COS cells, a cellular distribution that is clearly different from that seen for GFP-Rem in tsA201 cells (Fig. 1). Whether these differences are cell line-specific or dependent on the level of Rem expression is unclear. Mutations within the C terminus of RGK proteins that disrupt calmodulin binding have also been reported to promote nuclear translocation (12–14). Our data show that Rem-(1–265), Rem-(1–270), and Rem-(1–276) fail to bind calmodulin resin (Fig. 3B) and are not trafficked to the nucleus (Fig. 1C). However, the isolated Rem C terminus expressed as an RFP fusion protein is localized to punctate structures within the nucleus, suggesting that the Rem C terminus contains a cryptic nuclear localization sequence (Fig. 5C). Although both Rem^WT and Rem-(1–282) displayed robust Ca²⁺-dependent calmodulin binding and potent Ca²⁺ channel blockade (Fig. 3), Rem-(1–276) was shown to partially inhibit Ca²⁺ channel function, yet this mutant is incapable of binding calmodulin resin (Fig. 3B). Although these data indicate that calmodulin binding is not required for Rem-mediated Ca²⁺ channel regulation, it might more subtly modulate Rem activity. Thus, it will be important in future studies to evaluate the effect of calmodulin and 14-3-3 binding or site-selective phosphorylation within the polybasic domain to modulate RGK protein plasma membrane targeting.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Membrane-targeted Rem-(1–265) inhibits ICa. 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-RemWT. 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 RemWT and consistent with plasma membrane localization. C, TsA201 cells were transfected with plasmids expressing CaV1.2, CaV1b, 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 CaV1.2, CaV2a, 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 C. A significant difference (p < 0.05) between treatments is denoted by asterisks. F, currents at 5 mV from D. A significant difference (p < 0.05) between treatments is denoted by asterisks.

The observation that Ca_V subunit binding, unlike plasma membrane association, is not dependent upon the Rem C terminus suggests that binding and membrane localization are separable molecular events, and each may serve as an independent means of regulating Rem activity. To isolate the role of membrane trafficking from other functions of the C terminus, including PI lipid association (Fig. 2) and calmodulin binding (Fig. 3B) (14), we generated two chimeric Rem-(1–265) variants (Fig. 6) using the membrane targeting domains from K-Ras4B and H-Ras (25). Whereas the H-Ras CAAX domain relies upon prenylation/palmitoylation to direct membrane localization (25), the K-Ras4B region has many properties in common with Rem, including both calmodulin association and a polybasic domain capable of PI lipid-mediated PM targeting (23–25). Importantly, both anchors reconstituted plasma membrane association and partially restored Ca²⁺ channel regulation (Fig. 6), in agreement with recent studies examining Rem2 function using a similar strategy (15). Therefore, directing plasma membrane association appears to be the primary function of the Rem C terminus. However, since the chimeric proteins display more pronounced membrane trafficking (Fig. 6B) but do not fully recapitulate Rem-mediated Ca²⁺ channel inhibition (Fig. 6, C–F), it is likely that previously described interacting partners of and/or modifications to the Rem C terminus (including PI lipids, calmodulin, and/or 14-3-3 association, or protein kinase A/protein kinase C-mediated phosphorylation), although not essential for channel regulation, may contribute to Rem signaling (12–14, 18, 20, 23, 29–33).

In summary, we have found that the Rem C terminus serves as an essential targeting signal, probably acting through binding of the positively charged polybasic region to negatively charged PIP₂ and PIP₃ lipids, to direct Rem plasma membrane association. Although membrane localization and Ca_V subunit association are independent molecular events, we present strong evidence that both interactions play essential roles in Rem-mediated Ca²⁺ channel regulation. This new function for the conserved RGK C-terminal domain provides an opportunity for a variety of physiological pathways to influence RGK signaling. Clearly, additional studies will be needed to clarify the role of phosphatidylinositol lipid signaling and calmodulin/14-3-3 binding in both Rem trafficking and Ca²⁺ channel regulation.

	FOOTNOTES

 
^* This work was supported by Public Health Service Grants HL072936 (to D. A. A.), HL074091 (to J. S.), and P20 RR20171 from the National Center for Research Resources, National Institutes of Health (NIH) (to D. A. A.), an American Diabetes Association Junior Faculty award (to B. S. F.), and an American Heart Association predoctoral fellowship and NIH Interdisciplinary Cardiovascular Training Grant T32 HL072743 (to R. N. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ An Established Investigator of the American Heart Association.

² To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, BBSRB Rm. B-179, University of Kentucky College of Medicine, 741 S. Limestone St., Lexington, KY 40536-0509. Tel.: 859-257-6775; Fax: 859-323-1037; E-mail: dandres{at}uky.edu.

³ The abbreviations used are: RGK, Rem, Rem2, Rad, and Gem/Kir GTPases; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; WT, wild type; PI, phosphatidylinositol; RFP, red fluorescent protein; PtdIns, phosphatidylinositol; PIP, phosphorylated phosphatidylinositol; PIP2, diphosphorylated phosphatidylinositol; PIP3, PtdIns(3,4,5)P₃.

	ACKNOWLEDGMENTS

 
We thank Dr. Carole L. Moncman for expert assistance with the confocal imaging studies, Dr. Thomas C. Vanaman for the kind gift of calmodulin-Sepharose resin, and members of the Andres laboratory for critical reading of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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