Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase

Abstract

Rad/Rem/Gem/Kir (RGK) GTPases potently inhibit Ca(V)1 and Ca(V)2 (Ca(V)1-2) channels, a paradigm of ion channel regulation by monomeric G-proteins with significant physiological ramifications and potential biotechnology applications. The mechanism(s) underlying how RGK proteins inhibit I(Ca) is unknown, and it is unclear how key structural and regulatory properties of these GTPases (such as the role of GTP binding to the nucleotide binding domain (NBD), and the C-terminus which contains a membrane-targeting motif) feature in this effect. Here, we show that Rem inhibits Ca(V)1.2 channels by three independent mechanisms that rely on distinct configurations of the GTPase: (1) a reduction in surface density of channels is accomplished by enhancing dynamin-dependent endocytosis, (2) a diminution of channel open probability (P(o)) that occurs without impacting on voltage sensor movement, and (3) an immobilization of Ca(V) channel voltage sensors. The presence of both the Rem NBD and C-terminus (whether membrane-targeted or not) in one molecule is sufficient to reconstitute all three mechanisms. However, membrane localization of the NBD by a generic membrane-targeting module reconstitutes only the decreased P(o) function (mechanism 2). A point mutation that prevents GTP binding to the NBD selectively eliminates the capacity to immobilize voltage sensors (mechanism 3). The results reveal an uncommon multiplicity in the mechanisms Rem uses to inhibit I(Ca), predict new physiological dimensions of the RGK GTPase-Ca(V) channel crosstalk, and suggest original approaches for developing novel Ca(V) channel blockers.

Figure 1. Rem inhibits I_Ca by reducing both channel P_o and Q_max

A, top, exemplar whole-cell currents in a HEK 293 cell expressing recombinant Ca_V1.2 channel subunits (α_1C/β_2a). Bottom, exemplar current showing simultaneous isolation of tail (I_tail) and gating (I_gating) currents using a test pulse depolarization to the reversal potential (+40 mV). B, exemplar currents from cells co-expressing recombinant Ca_V1.2 channels and YFP–Rem. Same format as for A. Top inset, voltage protocol. C, exemplar currents from cells co-expressing recombinant Ca_V1.2 channels and YFP–Rem₂₆₅. Same format as for A. D, population peak current density (I_peak) versus voltage (V) relationship for cells expressing recombinant Ca_V1.2 channels (α_1C/β_2a) alone (grey circles, n= 7 for each point) or co-expressed with either YFP–Rem (triangles, n= 15 for each point) or YFP–Rem₂₆₅ (squares, n= 22 for each point). Data are means ±s.e.m.E, bar chart showing impact of Rem and Rem₂₆₅ on Q_max, *P < 0.05 when compared to Rem₂₆₅ using two-tailed unpaired t test. F, I_tail–Q_max scatter plot and regression line for Ca_V1.2 channels co-expressed with either YFP–Rem₂₆₅ (squares, slope = 6.98 ± 0.94 pA fC⁻¹, R²= 0.67) or YFP–Rem (triangles, slope = 1.6 ± 0.6 pA fC⁻¹, R²= 0.41). Open symbols are mean data for Ca_V1.2 +β_2a channels alone (circles) and in the presence of either YFP–Rem (triangles) or YFP–Rem₂₆₅ (squares). Inset, detail of scatter plots and linear fits near the origin.

Figure 2. Rem reduces surface density of Ca_V1.2 channels

A, cartoon showing strategy for detecting cell surface Ca_V1.2 channels with streptavidin-conjugated quantum dot (QD₆₅₅). B, top, confocal images of a HEK 293 cell expressing α_1C[BBS]–YFP alone. Middle, confocal images of a cell expressing α_1C[BBS]–YFP +β_2a. Bottom, images of a cell expressing α_1C[BBS]–YFP +β_2a+ CFP–Rem. The three channels detect CFP, YFP and QD₆₅₅ fluorescence signals, respectively. Scale bar is 2 μm, here and throughout. C–E, raw data from flow cytometry experiment showing the intensity of QD₆₅₅versus YFP signals for cells expressing α_1C[BBS]–YFP alone (C), α_1C[BBS]–YFP +β_2a (D) and α_1C[BBS]–YFP +β_2a+ CFP–Rem (E). 50,000 cells were counted for each condition. The vertical and horizontal lines represent threshold values arbitrarily set based on isochronal experiments using untransfected and single-colour control cells. Each dot represents a single cell. Dots have been arbitrarily colour-coded to facilitate visualization of distinct populations. Loosely, green dots represent α_1C[BBS]–YFP-positive cells that lack appreciable trafficking to the membrane (low QD₆₅₅ signal), while red dots represent α_1C[BBS]–YFP-positive cells that display robust Ca_V1.2 channel trafficking to the surface (high QD₆₅₅ signal). Grey dots in the bottom left quadrant correspond to untransfected cells. F, bar chart showing normalized mean QD₆₅₅ fluorescence signals across separate flow cytometry experiments, n= 5 for each condition. Data were normalized to mean QD₆₅₅ signals from cells expressing α_1C[BBS]–YFP +β_2a+ CFP–Rem₂₆₅ in isochronal experiments. *Significantly different (P < 0.05) from α_1C[BBS]–YFP +β_2a using one-way ANOVA and Bonferroni post hoc means comparisons. G, normalized α_1C[BBS]–YFP fluorescence signals in the same analyses window used to compute QD₆₅₅ signals in F. Data were normalized to mean YFP fluorescence signals from cells expressing α_1C[BBS]–YFP +β_2a+ CFP–Rem₂₆₅. H, population I_peak–V relationship for channels reconstituted with α_1C[BBS]–YFP +β_2a in the presence of either CFP–Rem₂₆₅ (black squares, n= 5 for each point) or CFP–Rem (red triangles, n= 5 for each point).

Figure 3. Dominant negative dynamin reverses Rem-induced decrease in Ca_V1.2 channel surface density

A, confocal images of cells expressing α_1C[BBS]–YFP + DNM– (top row), α_1C[BBS]–YFP +β_2a+DNM– (middle), and α_1C[BBS]–YFP +β_2a+ CFP–Rem +DNM– (bottom). B, normalized mean QD₆₅₅ fluorescence. Data were normalized to mean QD₆₅₅ signals from α_1C[BBS]–YFP +β_2a+ DNM– cells in isochronal experiments. *P < 0.05 compared to α_1C[BBS]–YFP +β_2a+ DNM– using two-tailed unpaired t test. C, population I_peak–V relationship for channels reconstituted with α_1C[BBS]–YFP +β_2a+ Rem₂₆₅+ DNM– (squares, n= 5 for each point) or α_1C[BBS]–YFP +β_2a+ Rem + DNM– (triangles, n= 5 for each point). D, impact of DNM– on Q_max from cells expressing Ca_V1.2 in the presence of either Rem₂₆₅ or Rem, *P < 0.05 when compared to Rem₂₆₅+ DNM– using two-tailed unpaired t test.

Figure 4. Role of Rem GTP binding in I_Ca inhibition

A, cartoon showing canonical regulation of monomeric GTPases. Proteins cycle between an inactive GDP-bound and an active GTP-bound state. GTP–GDP exchange and GTPase reactions are catalysed by guanine nucleotide exchange factors (GEF) and GTPase activating proteins (GAP), respectively. B, left, exemplar current in control (YFP–Rem₂₆₅) cells showing simultaneous isolation of I_tail and I_gating using a test pulse depolarization to V_rev. Right, exemplar I_tail and I_gating in cells co-expressing recombinant Ca_V1.2 channels and YFP–Rem[T94N]. C, population I_peak–V relationships for isochronal cells expressing Ca_V1.2 channels together with either YFP–Rem₂₆₅ (squares, n= 8 for each point) or YFP–Rem[T94N] (triangles, n= 10 for each point). Data are means ±s.e.m. D, I_tail–Q_max scatter plot for Ca_V1.2 channels in the presence of either YFP–Rem₂₆₅ (squares, slope = 8.9 ± 1.9 pA fC⁻¹, R²= 0.65) or YFP–Rem[T94N] (triangles, slope = 1.15 ± 0.53 pA fC⁻¹, R²= 0.23). E, bar chart showing lack of effect of YFP–Rem[T94N] on Q_max. F, normalized mean QD₆₅₅ fluorescence, *P < 0.05 compared to Rem[T94N]+ DNM– using two-tailed unpaired t test.

Figure 5. Role of the Rem C-terminus extension in I_Ca inhibition

A, exemplar currents from a cell expressing recombinant Ca_V1.2 channels and CFP–Rem₂₆₅–C1_PKCγ in the absence (left) or presence (right) of 1 μm PdBu. Inset, confocal images showing that PdBu translocates CFP–Rem₂₆₅–C1_PKCγ from the cytosol to plasma and nuclear membranes. B, diary plot showing time course and relative effects of PdBu (red symbols) on normalized I_tail (black squares) and Q_max (black triangles). C, population I_peak–V plot in cells co-expressing recombinant Ca_V1.2 channels and CFP–Rem₂₆₅–C1_PKCγ in the absence (black squares, n= 6 for each point) or presence (red circles, n= 6) of 1 μm PdBu. D, bar charts showing the relative effects of PdBu on I_tail (left) and Q_max (right). Data are means ±s.e.m. *P < 0.05 compared to control (no PdBu) using two-tailed paired t test. E, confocal images of HEK 293 cells expressing recombinant Ca_V1.2 channels with either CFP–Rem₂₆₅ (left) or CFP–Rem₂₆₅–C1_PKCγ (right) in the absence and presence of PdBu. F, normalized mean QD₆₅₅ fluorescence.

Figure 6. Non-membrane targeted Rem derivatives that inhibit I_Ca

A, confocal images showing subcellular localization of YFP–Rem[L271G] and CFP–β₃. B, exemplar whole-cell currents in a HEK 293 cell expressing recombinant Ca_V1.2 channels and YFP–Rem[L271G]. C, population I_peak–V plots for cells expressing recombinant Ca_V1.2 channels and either YFP–Rem₂₆₅ (black squares, n= 15 for each point) or YFP–Rem[L271G] (red triangles, n= 9 for each point). Data are means ±s.e.m. D, I_tail–Q_max scatter plots and regression lines for Ca_V1.2 channels co-expressed with either YFP–Rem₂₆₅ (black squares, slope = 5.73 ± 1.05 pA pF⁻¹, R²= 0.73) or YFP–Rem[L271G] (red triangles, slope = 4.31 ± 1.49 pA fC⁻¹, R²= 0.38). E–H and I–L, data for NLS–YFP–Rem[L271G] (n= 15 for each point) and NES–YFP–Rem[L271G] (n= 15 for each point), respectively. Inset, detail of scatter plots and linear fits near the origin. E–H and I–L, same format as for A–D. For NLS–YFP–Rem[L271G] (slope = 5.48 ± 1.48 pA fC⁻¹, R²= 0.56), and for NES–YFP–Rem[L271G] (slope = 0.08 ± 0.37 pA fC⁻¹). Data for control cell expressing YFP–Rem₂₆₅ (black squares) is identical to those in C and D. M, bar chart showing the impact of various Rem derivatives on Q_max. *Significantly different (P < 0.05) from Rem₂₆₅ using one-way ANOVA and Bonferroni post hoc analyses. N, Left, confocal images of a cell expressing α_1C[BBS]–YFP +β_2a+ NES–CFP–Rem[L271G]. Right, normalized mean QD₆₅₅ fluorescence. *P < 0.05 compared to control cells expressing α_1C[BBS]–YFP +β_2a using two-tailed unpaired t test.

Figure 7. Mechanisms underlying Rem inhibition of I_Ca

In control, unmodified channels, channels respond to depolarization by undergoing voltage-dependent closed–closed (C–C) transitions during which the voltage sensors move, followed by a voltage-independent conformational change that opens the channel (C–O transition). Rem constitutes a ‘triple threat’ to Ca_V1.2 channels, inhibiting I_Ca by three distinct, functionally redundant mechanisms that rely on different configurations of the GTPase. All three mechanisms are assumed to rely on the formation of a ternary α₁–β–Rem complex since Rem does not disrupt the Ca_V channel α₁–β subunit interaction. (I) Rem reduces surface density of Ca_V1.2 channels by enhancing dynamin-dependent endocytosis. This mechanism requires the Rem C-terminus and nucleotide-binding domain, but is GTP-independent; (II) Rem diminishes P_o without affecting voltage sensor movement (C–C transitions are unaffected, while forward equilibrium for the C–O transition is reduced). Membrane targeting of the NBD by either the C-terminus or a generic membrane-targeting module is sufficient for this effect, whereas GTP binding to the NBD is not necessary (represented as a red hexagon). (III) Rem immobilizes Ca_V1.2 channel voltage sensors (forward equilibria for C–C transitions are reduced). This mechanism requires the C-terminus and GTP bound to the nucleotide-binding domain of Rem (represented by green hexagon).