Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density

Abstract

Rad, Gem/Kir, Rem, and Rem2 are members of the Ras-related RGK (Rad, Gem, and Kir) family of small GTP-binding proteins. Heterologous expression of RGK proteins interferes with de novo calcium channel assembly/trafficking and dramatically decreases the amplitude of currents arising from preexisting high-voltage-activated calcium channels. These effects probably result from the direct interaction of RGK proteins with calcium channel beta subunits. Among the RGK family, Rem2 is the only member abundantly expressed in neuronal tissues. Here, we examined the ability of Rem2 to modulate endogenous voltage-activated calcium channels in rat sympathetic and dorsal root ganglion neurons. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high-voltage-activated calcium channels without affecting low-voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels required both the Ras homology (core) domain and the polybasic C terminus. Mutation of a putative GTP/Mg2+ binding motif in Rem2 did not affect suppression of calcium currents. Loading neurons with GDP-beta-S via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally, [(125)I]Tyr22-omega-conotoxin GVIA cell surface binding in tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when calcium current was totally abolished. Together, our results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel beta subunits in the preassembled N-type channel, thereby forming a nonconducting species.

Figure 1.

Effects of heterologous expression of Rem2 on N-type Ca²⁺ channels of sympathetic neurons. A, Superimposed representative I_Ca traces recorded from a control (uninjected) neuron (left) and a neuron expressing Rem2 (right). Expression was accomplished with nuclear microinjection (see Materials and Methods). I_Ca was evoked by a 70 ms step to the indicated voltages from a holding potential of -80 mV. B, Average ± SEM I_Ca I-V relationships for I_Ca recorded from control neurons (open circles) and neurons expressing Rem2 (filled circles) or Rem2t (open triangles). I_Ca density was calculated from I_Ca amplitude at a series of test pulses normalized to cell membrane capacitance. C, Average ± SEM I_Ca density in control neurons (open bar) and neurons expressing GFP, Rem2, Rem2t, GFP-Rem2t, or Rem2t-GFP as indicated (filled bars). I_Ca density was calculated from I_Ca amplitude at a test pulse of +10 mV normalized to cell membrane capacitance. D, The mean values from B were scaled to facilitate comparison of the current density-voltage relationship. E, Superimposed I_Ca traces recorded in the absence or presence of NE (10 μm) from a control neuron (left) and a neuron expressing Rem2 (right). I_Ca was evoked at 0.1 Hz with the voltage protocol illustrated. F, Average I_Ca inhibition mediated by NE in control neurons and neurons expressing Rem2 or Rem2t. The number of neurons tested is shown in parentheses. ^***p < 0.001 versus controls, ANOVA followed by Dunnett's test. Uninj., Uninjected.

Figure 2.

Treatment of sympathetic neurons with cycloheximide, a protein translation blocker, did not alter I_Ca amplitude. A, Representative whole-cell I_Ca traces from a control neuron and a neuron pretreated with cycloheximide (0.35 mm) for ∼24 h. The voltage protocol above the current traces was repeated at 0.1 Hz. B, Average ± SEM I_Ca amplitude and density in control neurons (open bar) and neurons pretreated with cycloheximide (filled bar) for 22-29 h. C, Phase-contrast (left column) and fluorescent (right column) micrographs illustrating adenovirus-mediated GFP expression in control neurons (top row) and neurons treated with cycloheximide (bottom row).

Figure 3.

Heterologously expressed Rem2 is enriched at the plasma membrane in hippocampal neurons and COS-7 cells. Fluorescent micrographs were taken from hippocampal neurons (top row) and COS-7 cells (bottom row) ∼24 h after transfection of cDNA constructs encoding GFP-Rem2t (left column) or Rem2t-GFP (right column). To facilitate the visualization of GFP fluorescence at plasma membrane areas, COS-7 cells were briefly treated with PBS containing trypsin-EDTA to cause cell rounding.

Figure 4.

The structure-function relationship of Rem2. A, Average ± SEM I_Ca density in uninjected neurons (open bar) and neurons injected (filled bars) with cDNA constructs encoding one of the truncated or recombinant Rem2 (middle panel) as illustrated in the schematic diagram (left panel). Average ± SEM I_Ca density in uninjected neurons (open bar) and neurons injected (filled bars) with the corresponding GFP-fusion cDNA constructs were summarized in the right panel. I_Ca density was calculated from I_Ca amplitude at a test pulse of +10 mV normalized to cell membrane capacitance. ^***p < 0.001 versus control neurons, ANOVA. B, Fluorescent micrographs were taken from HEK293 cells ∼24 h after transfection with the cDNA construct as indicated above each graph. C, Fluorescent micrographs were taken from sympathetic neurons ∼20 h after intranuclear injection with the cDNA construct as indicated above each graph. Gi1-10aa, The first 10 residues of Gα_i1; K-ras4B-c-tail, the last 23 residues at the C-end of K-ras4B; Uninj., uninjected.

Figure 5.

GTP binding may not be required for Rem2-mediated downregulation of I_Ca in sympathetic neurons. A, Average ± SEM I_Ca density in control neurons (open bar) and neurons expressing (filled bars) Rem2t-S129N or GFP-Rem2t-S129N. I_Ca density was calculated as in Figure 1C. ^***p < 0.001 versus control neurons, ANOVA. B, Fluorescent micrographs were taken from COS-7 cells ∼24 h after transfection with GFP-Rem2t or GFP-Rem2t-S129N cDNA constructs as indicated. C, Comparison of NE-mediated inhibition of I_Ca recorded with pipette solutions containing either GTP (left) or GDP-β-S (right). I_Ca was evoked as in Figure 2 A. D, Time course of I_Ca recorded from a neuron expressing Rem2t. GDP-β-S (2 mm) was included in the patch-pipette solution. Superimposed I_Ca traces recorded at 30s and 20 min after patch rupture. E, Mean ± SEM I_Ca density at 30 s and 10 min after the establishment of whole-cell recording (2 mm GDP-β-S was included in the pipette solution) in control neurons and neurons expressing Rem2t or Rem2. ^*p < 0.05, paired t test. Uninj., Uninjected; con, control.

Figure 6.

Expression of GFP-Rem2t in DRG neurons attenuated HVA but spared LVA I_Ca. A, I_Ca traces recorded from two separate DRG neurons infected with Ad-GFP. The left panel illustrates a neuron with a large LVA I_Ca component. The right panel illustrates a neuron with negligible LVA I_Ca. The voltage protocol used to evoke I_Ca is illustrated above the traces. The current evoked at -40 mV arises from LVA Ca²⁺ channels, whereas the current evoked at +10 mV arises primarily from HVA Ca²⁺ channels (see Materials and Methods). To facilitate analysis, DRG neurons were divided into a group displaying a high density of LVA I_Ca (>15 pA/pF; left) and a group displaying virtually no LVA I_Ca (< 3pA/pF; right). Bar graphs summarizing the average I_Ca density arising from LVA (black) and HVA (gray) channels 36-48 h after infection with Ad-GFP. B, The same analysis as shown in A for neurons infected with adenoviruses expressing GFP-Rem2t. LVA I_Ca amplitude was measured 15 ms after initiation of the voltage step to -40 mV, whereas HVA I_Ca amplitude was measured 10 ms from the start of the voltage to +10 mV. I_Ca density was calculated from I_Ca amplitude normalized to cell membrane capacitance. Number of neurons tested is shown in parentheses.

Figure 7.

Heterologous expression of Rem2 in tsA201 cells stably expressing Ca_vα₁2.2/β3/α₂δ abolished I_Ca without affectingω-conotoxin GVIA binding. A, Bar graphs summarizing the average I_Ca density of cells transfected 19-53 h earlier with cDNA constructs encoding GFP, GFP-Rem2t, or GFP-Rem2. I_Ca density was calculated as in Figure 1C. The number of cells tested is shown in parentheses. B, Comparison of [¹²⁵I]Tyr²²-ω-conotoxin GVIA binding to different numbers of tsA201 cells expressing Ca²⁺ channels. To compensate for potential nonspecific binding, HEK293 cells were added to maintain a total of 2 × 10⁵ cells per well. After overnight culture, cells were incubated with [¹²⁵I]Tyr²²-ω-conotoxin GVIA (1 nm) for 40 min and washed thoroughly, and the bound ligand was quantified with a scintillation counter. C, Mean [¹²⁵I]Tyr²²-ω-conotoxin GVIA bound to tsA201 cells 26 h after transfection with cDNA constructs encoding GFP, GFP-Rem2t, or GFP-Rem2. D, Transfection efficiency (measured as the percentage of cells displaying GFP fluorescence) was determined from parallel experiments. A total of 500 cells were examined for each construct by phase contrast and epifluorescence microscopy (40× objective, 0.60 numerical aperture).