Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

Akil A. Puckerin; Donald D. Chang; Zunaira Shuja; Papiya Choudhury; Joachim Scholz; Henry M. Colecraft

doi:10.1073/pnas.1811024115

Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved October 8, 2018 (received for review June 28, 2018)

Significance

Influx of calcium ions through surface membrane calcium channels that open in response to electrical signals is important for vital biological processes including generation of the heartbeat and nerve cell communication. Blocking such calcium channels in a tissue- and isoform-specific manner is a sought-after treatment strategy for diseases including chronic pain and Parkinson’s disease. Proteins that can be expressed in cells to selectively block different calcium channel types have particular advantages over conventional small-molecule blockers. A four-member family of proteins known as RGK proteins strongly inhibit calcium channels, but do so in a non-selective manner, limiting their potential usefulness. Here we identified mutated RGK proteins that perform as isoform-selective calcium channel blockers, advancing the therapeutic potential of these proteins.

Abstract

Genetically encoded inhibitors for voltage-dependent Ca²⁺ (Ca_V) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca²⁺ (Ca_V1/Ca_V2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary Ca_Vβ-subunits. To investigate latent Ca_Vβ-independent components of inhibition, we coexpressed each RGK individually with Ca_V1 (Ca_V1.2/Ca_V1.3) or Ca_V2 (Ca_V2.1/Ca_V2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β_2a or a mutant version (β_2a,TM) that does not bind RGKs. All four RGKs strongly inhibited Ca_V1/Ca_V2 channels reconstituted with WT β_2a. By contrast, when channels were reconstituted with β_2a,TM, Rem inhibited only Ca_V1.2, Rad selectively inhibited Ca_V1.2 and Ca_V2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT Ca_Vβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted Ca_V1.2 while Rad[R208A/L235A] inhibited Ca_V1.2/Ca_V2.2 but not Ca_V1.3/Ca_V2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous Ca_V1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for Ca_V1.2 and Ca_V1.2/Ca_V2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific Ca_V1/Ca_V2 channels to develop selective GECCIs with properties unmatched by current small-molecule Ca_V channel blockers.

High voltage-activated (HVA) Ca²⁺ (Ca_V) channels convert electrical signals into Ca²⁺ influx that controls myriad essential processes including neuronal communication, muscle contraction, hormone release, and activity-dependent gene transcription (1). HVA Ca_V channels are composed of a pore-forming α₁- assembled with auxiliary β-, α₂δ-, and γ-subunits and calmodulin. There are seven α₁-subunits (Ca_V1.1 to 1.4 and Ca_V2.1 to 2.3), four Ca_Vβs (β₁ to β₄), and three α₂δs (α₂δ1 to α₂δ3), each with multiple splice variants. Ca_Vα₁-subunits contain the voltage sensor, selectivity filter, and channel pore, while auxiliary subunits regulate channel properties—Ca_Vβs are obligatory for α₁-trafficking to the plasma membrane and for modulating channel gating (2); α₂δs enhance channel surface trafficking and also modulate channel gating (3); and calmodulin promotes channel trafficking, enhances basal open probability (P_o), and confers feedback Ca²⁺ regulation of channel gating (4, 5).

Ca_V channels are also regulated by various intracellular signaling proteins and posttranslational modifications as a mechanism to control physiology. Pharmacological blockade of Ca_V1/Ca_V2 channels is an important treatment strategy for diverse diseases including hypertension, cardiac arrhythmias, chronic pain, and Parkinson’s disease (1). RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind Ca_Vβ-subunits and profoundly inhibit all Ca_V1/Ca_V2 channels (6 ⇓–8). Given their properties, RGKs straddle two worlds with respect to their impact on Ca_V1/Ca_V2 channels—they are (i) potentially powerful physiological regulators by virtue of their capacity to tune intracellular Ca²⁺ signals, and (ii) prototype genetically encoded Ca_V channel blockers with possible therapeutic and biotechnological applications (9). Consistent with important physiological roles, Rad knockout mice exhibit increased cardiac Ca_V1.2 currents and cardiac hypertrophy, while Gem-deficient mice display glucose intolerance and impaired glucose-stimulated insulin release. Regarding their use as potential therapeutics, expression of Gem in the atrioventricular node was effective at electrically uncoupling ventricular excitation from the fibrillating atria in a porcine model of atrial fibrillation (10). A Rem derivative engineered to selectively target and inhibit caveola-localized Ca_V channels effectively inhibited pacing-induced NFATc3-GFP translocation to the nucleus in adult feline ventricular cardiomyocytes, without affecting excitation–contraction coupling (11).

A major limitation for the use of RGKs as genetically encoded Ca_V channel blockers involves their lack of selectivity for particular Ca_V1/Ca_V2 isoforms. Rem inhibits Ca_V1.2 channels using multiple mechanisms including reduced channel surface density, diminished P_o, and partial immobilization of voltage sensors (12). At least one of these mechanisms (decreased P_o) involves the simultaneous association of Rem with the auxiliary Ca_Vβ-subunit and the plasma membrane (13, 14). This mechanism likely accounts for the indiscriminate nature of RGK inhibition of Ca_V1/Ca_V2 channels, since all four RGKs bind Ca_Vβ-subunits and the plasma membrane, and Ca_Vβs are obligatory for forming functional channels. Beyond the β-binding mechanism, we previously showed that Rem can also inhibit Ca_V1.2 channels by directly binding to the pore-forming α_1C-subunit (15). Potentially, such an α₁-subunit–dependent mechanism could be exploited to develop genetically encoded Ca_V1/Ca_V2 isoform-selective inhibitors. Several outstanding questions need to be addressed to realize this potential. Does Rem inhibit other Ca_V1/Ca_V2 channels beyond Ca_V1.2 in a β-binding–independent manner? Do other RGKs beyond Rem inhibit Ca_V1/Ca_V2 channel isoforms in a β-binding–independent manner? Are both β-binding–dependent and β-binding–independent mechanisms of RGK inhibition of particular Ca_V1/Ca_V2 channels prevalent in native excitable cells? If so, do the two modes of inhibition display physiologically meaningful differences?

Here, focusing on four Ca_V channels (Ca_V1.2, Ca_V1.3, Ca_V2.1, and Ca_V2.2), we show that Rem uniquely blocks Ca_V1.2 using a β-binding–independent mechanism. Consistent with this finding, a mutant Rem that cannot bind β (Rem[R200A/L227A]) selectively blocked Ca_V1.2, with no effect on the closely related Ca_V1.3 channel. Further, Rad inhibited Ca_V1.2 and Ca_V2.2 (but not Ca_V1.3 or Ca_V2.1) channels via a β-binding–independent mechanism. Accordingly, a β-binding–deficient Rad mutant (Rad[R208A/L235A]) effectively blocked Ca_V1.2/Ca_V2.2, but not Ca_V1.3/Ca_V2.1, channels. Both Rem[R200A/L227A] and Rad[R208A/L235A] strongly inhibited endogenous Ca_V1.2 channels in adult ventricular cardiomyocytes. Finally, Rem[R200A/L227A] and Rad[R208A/L235A], but not Gem[R196A/V223A], inhibited HVA Ca_V channels in somatosensory dorsal root ganglion (DRG) neurons, albeit with different magnitudes reflecting their selectivity for either Ca_V1.2 alone or Ca_V1.2/Ca_V2.2, respectively. Altogether, we have exploited latent β-binding–independent inhibition of Ca_V1.2 and Ca_V1.2/Ca_V2.2 channels by Rem and Rad, respectively, to engineer genetically encoded isoform-selective Ca_V channel blockers.

Results

Differential Prevalence of β-Binding–Dependent and β-Binding–Independent Rem Inhibition Across Distinct Ca_V1/Ca_V2 Channels.

We profiled β-binding–dependent (BBD) and β-binding–independent (BBI) Rem inhibition of Ca_V channels by reconstituting distinct pore-forming α₁-subunits with either wild-type β_2a or a mutant β_2a (β_2a,TM) that does not bind RGKs. HEK293 cells expressing α_1C + β_2a expressed robust Ba²⁺ currents (I_Ba) that were virtually eliminated when Rem was coexpressed (Fig. 1 A and B and SI Appendix, Fig. S1). Similarly, cells expressing α_1C + β_2a,TM displayed I_Ba that was significantly inhibited by Rem (Fig. 1 A and B and SI Appendix, Fig. S1), indicating the incidence of both BBD and BBI Rem inhibition of Ca_V1.2 channels. These results confirm our previous report that both BBD and BBI mechanisms contribute to Rem inhibition of Ca_V1.2 (15). I_Ba influx through reconstituted Ca_V1.3 channels (α_1D + β_2a) was eliminated by Rem. However, Ca_V1.3 channels reconstituted with α_1D + β_2a,TM were refractory to Rem (Fig. 1 C and D and SI Appendix, Fig. S1), indicating the absence of BBI inhibition, and revealing a fundamental difference from Ca_V1.2. Similar to Ca_V1.3, Ca_V2.1 (Fig. 1 E and F and SI Appendix, Fig. S1) and Ca_V2.2 (Fig. 1 G and H and SI Appendix, Fig. S1) channels were inhibited by Rem only when reconstituted with WT β_2a, but not β_2a,TM, a modulation profile consistent with exclusively BBD inhibition.

Fig. 1.

Rem uniquely inhibits Ca_V1.2 using both β-binding–dependent and β-binding–independent mechanisms. (A) Exemplar Ca_V1.2 Ba²⁺ currents elicited from HEK293 cells expressing α_1C + β_2a ± Rem (columns 1 and 2) or α_1C + β_2a,TM ± Rem (columns 3 and 4). Ba²⁺ currents were elicited by 25-ms test pulse depolarizations (from −50 to +100 mV in 10-mV increments) from a holding potential of −90 mV. (B) Population bar charts showing the impact of Rem on peak I_Ba from channels reconstituted with either α_1C + β_2a (Left) or α_1C + β_2a,TM (Right). *P < 0.01, Student’s unpaired t test. (C and D) Data for Ca_V1.3 channels reconstituted with either α_1D + β_2a ± Rem or α_1D + β_2a,TM ± Rem, same format as A and B. (E and F) Data for Ca_V2.1 channels reconstituted with either α_1A + β_2a ± Rem or α_1A + β_2a,TM ± Rem, same format as A and B. (G and H) Data for Ca_V2.2 channels reconstituted with either α_1B + β_2a ± Rem or α_1B + β_2a,TM ± Rem, same format as A and B. Data are means ± SEM.

Engineering a Ca_V1.2-Selective Inhibitor from Rem.

The finding that BBI Rem inhibition of I_Ba is a unique property of Ca_V1.2 suggested the possibility of engineering a Ca_V1.2-selective genetically encoded inhibitor by generating a Rem mutant that does not bind Ca_Vβ. A previous mutagenesis study identified residues in RGKs that were critical for their interaction with Ca_Vβs but did not disrupt their tertiary structure, as evaluated by GTP/GDP binding assays (16). Based on these findings, we introduced two point mutations (R200A, L227A) into Rem and used FRET to evaluate the association of Rem[R200A/L227A] with Ca_Vβ (Fig. 2A). HEK293 cells coexpressing CFP-WT Rem + YFP-β₃ displayed a significantly elevated FRET (FRET efficiency 0.188 ± 0.006, n = 127) compared with negative control cells expressing CFP-FRB + β₃-YFP (FRET efficiency 0.046 ± 0.002, n = 126) (Fig. 2B), consistent with well-known Rem–Ca_Vβ interaction (6, 7). By comparison, cells coexpressing CFP-Rem[R200A/L227A] + β₃-YFP displayed a markedly lower FRET (FRET efficiency 0.058 ± 0.002, n = 138) that did not differ from control cells, consistent with reduced protein interaction (Fig. 2B). Additional insights into the relative affinities of Rem and Rem[R200A/L227A] for Ca_Vβ₃ was provided from binding analyses of FRET efficiency vs. A_free scatterplots (Fig. 2C), which indicated a fivefold decreased affinity of Rem[R200A/L227A] for Ca_Vβ₃ compared with WT Rem (Fig. 2C).

Fig. 2.

Rem[R200A/L227A] does not bind Ca_Vβ and selectively inhibits Ca_V1.2. (A) Crystal structure of Rem G domain with residues R200 and L227 highlighted. (B) FRET efficiency measurements in HEK293 cells coexpressing CFP-FRB + β₃-YFP (control), CFP-Rem + β₃-YFP, or CFP-Rem[R200A/L227A] + β₃-YFP. Data are means ± SEM. *P < 0.01, one-way ANOVA. (C) Binding-curve analyses of FRET experiments. (D) Population I_peak–V plots for cells expressing α_1C + β_2a (black squares; n = 9), α_1C + β_2a + Rem[L200A/L227A] (red squares; n = 10); α_1D + β_2a (black circles; n = 11), α_1D + β_2a + Rem[L200A/L227A] (red circles; n = 13); α_1A + β_2a (black triangles; n = 10), α_1A + β_2a + Rem[L200A/L227A] (red triangles; n = 12); and α_1B + β_2a (black diamonds; n = 11), α_1B + β_2a + Rem[L200A/L227A] (red diamonds; n = 12). *P < 0.05, Student’s t test. (E) Exemplar cultured adult cardiomyocytes expressing GFP (Top) or CFP-Rem[L200A/L227A] (Bottom). (F) Representative Ba²⁺ currents from adult guinea pig ventricular cardiomyocytes expressing either GFP (Left; control) or CFP-Rem[L200A/L227A] (Right). (G) Population I_peak–V plots for cardiomyocytes expressing GFP (black squares; n = 8) or CFP-Rem[L200A/L227A] (red triangles; n = 10).

We next determined whether Rem[R200A/L227A] would function as a Ca_V1.2-selective inhibitor as hypothesized. Indeed, HEK293 cells coexpressing recombinant Ca_V1.2 (α_1C + β_2a) channels and Rem[R200A/L227A] displayed significantly lower I_Ba compared with control cells expressing Ca_V1.2 alone (Fig. 2D; I_peak,10mV = 62.3 ± 14.3 pA/pF, n = 9 for α_1C + β_2a compared with I_peak,10mV = 24.9 ± 4.9 pA/pF, n = 10 for α_1C + β_2a + Rem[R200A/L227A]; P = 0.0194, Student’s t test; SI Appendix, Fig. S2). In sharp contrast, recombinant Ca_V1.3, Ca_V2.1, and Ca_V2.2 were refractory to Rem[R200A/L227A] (Fig. 2D and SI Appendix, Fig. S2), consistent with this engineered protein being a Ca_V1.2-selective blocker. The finding that Ca_V2.2 is not inhibited by a β-binding–deficient Rem recapitulates a previous similar finding by Beqollari et al. (17).

To determine whether Rem[R200A/L227A] could inhibit endogenous Ca_V1.2 channels, we assessed its efficacy in blocking I_Ba conducted through native Ca_V1.2 channels in guinea pig ventricular cardiomyocytes. We generated adenovirus enabling robust expression of YFP-Rem[R200A/L227A] (Fig. 2E). Compared with control cells expressing GFP, cardiomyocytes expressing YFP-Rem[R200A/L227A] displayed a significantly reduced I_Ba at all test voltages (Fig. 2 F and G; I_peak,0mV = 22.6 ± 4.6 pA/pF, n = 8 for GFP compared with I_peak,0mV = 9.1 ± 2.3 pA/pF, n = 10, for YFP-Rem[R200A/L227A]), thus demonstrating BBI Rem inhibition of endogenous Ca_V1.2 channels in the heart.

Prevalence of BBD and BBI RGK Inhibition Across the Ca_V1/Ca_V2 Channel Family.

We wondered whether other RGKs display BBI inhibition of Ca_V1/Ca_V2 channels that could be similarly exploited to generate selective genetically encoded inhibitors for Ca_V channels (GECCIs). We profiled the occurrence of BBD and BBI inhibition across RGKs and Ca_V1/Ca_V2 channels by assessing the impact of Gem, Rad, and Rem2 on recombinant Ca_V channels reconstituted with either WT β_2a or β_2a,TM (Fig. 3A and SI Appendix, Fig. S3). Ca_V1.3 channels reconstituted with WT β_2a (α_1D + β_2a) were uniformly inhibited by Gem, Rad, and Rem2, respectively (SI Appendix, Fig. S3B). By contrast, these three RGKs had no impact on I_Ba influx through α_1D + β_2a,TM channels (SI Appendix, Fig. S3B). Together, these results indicate that all RGKs inhibit Ca_V1.3 channels solely through a BBD mechanism. We obtained virtually identical results with reconstituted Ca_V2.1 channels—α_1A + β_2a channels were inhibited by Gem, Rad and Rem2, whereas α_1A + β_2a,TM channels were refractory to these RGKs (SI Appendix, Fig. S3C). Hence, Ca_V2.1 channels also display exclusively BBD RGK inhibition. The finding that Rem2 inhibits Ca_V2.1 in a solely BBD manner agrees with a previous result showing that Rem2 abolishes current through Ca_V2.1 channels reconstituted with WT β₄ but not a mutant β₄ lacking the capacity to bind Rem2 (18). Our finding that Gem requires binding to Ca_Vβ to decrease Ca_V2.1 is in disagreement with a previous report that Gem binding to Ca_Vβ₃ was not necessary for its capacity to inhibit Ca_V2.1 current (19). The reasons for this discrepancy are unclear, though one possibility is the intrinsic differences between Xenopus oocytes (used in the previous study) and the mammalian cells used here. As expected, wild-type Ca_V2.2 channels (α_1B + β_2a) were robustly inhibited by Gem, Rad, and Rem2, respectively. Interestingly, while channels reconstituted with α_1B + β_2a,TM were unaffected by Gem and Rem2, they were significantly inhibited by Rad (Fig. 3B). Therefore, Rad uniquely mediates both BBD and BBI inhibition of Ca_V2.2 channels. We previously reported that Rad (but not Gem or Rem2) also supports BBD and BBI inhibition of Ca_V1.2 (15). Together, these reports suggested that eliminating Rad binding to Ca_Vβ would generate a selective inhibitor of Ca_V1.2/Ca_V2.2 channels.

Fig. 3.

Prevalence of β-binding–dependent and β-binding–independent RGK inhibition of I_Ba in Ca_V2.2 channels. (A) Schematic of HVA Ca_V channel pore-forming α₁-subunit binding to β_2a or β_2a,TM with putative binding sites responsible for β-binding–dependent (solid arrow) and β-binding–independent (dashed arrows) RGK inhibition of current. (B) Bar charts showing impact of Gem, Rad, and Rem2 on Ca_V2.2 channels reconstituted with either α_1B + β_2a (Left) or α_1B + β_2a,TM (Right). Data are means ± SEM. *P < 0.05 compared with control, one-way ANOVA.

Engineering a Ca_V1.2- and Ca_V2.2-Selective Inhibitor from Rad.

Using an approach similar to the generation of Rem[R200A/L227A], we introduced equivalent mutations in Rad to create Rad[R208A/L235A]. Three-cube FRET experiments confirmed that cells expressing CFP-Rad[R208A/L235A] + YFP-β₃ showed lower FRET efficiency (0.051 ± 0.002, n = 142) compared with CFP-Rad + YFP-β₃ (0.123 ± 0.004, n = 174) (Fig. 4B). Binding-curve analyses indicated an eightfold decrease in affinity of CFP-Rad[R208A/L235A] for YFP-β₃ compared with CFP-Rad (Fig. 4C). As predicted, Rad[R208A/L235A] significantly inhibited currents through recombinant Ca_V1.2 (α_1C + β_2a) and Ca_V2.2 (α_1B + β_2a) channels but had no impact on either Ca_V1.3 (α_1D + β_2a) or Ca_V2.1 (α_1A + β_2a) channels (Fig. 4D and SI Appendix, Fig. S4). Hence, Rad[R208A/L235A] is a Ca_V1.2/Ca_V2.2-selective inhibitor. When expressed in guinea pig ventricular cardiomyocytes, Rad[R208A/L235A] inhibited endogenous Ca_V1.2 channels to almost the same extent as WT Rad (Fig. 4 E and F), revealing a strong BBI Rad inhibition of Ca_V1.2 in the heart. It was previously shown that Rad-inhibited Ca_V1.2 channels are not up-regulated by activated protein kinase A (PKA) (20). We found that I_Ba through ventricular Ca_V1.2 channels inhibited by Rad[R208A/L235A] was robustly increased by 1 μM forskolin, in sharp contrast to the lack of modulation observed with WT Rad-inhibited channels (Fig. 4 G and H and SI Appendix, Fig. S5). Hence, cardiac Ca_V1.2 channels undergoing either BBD or BBI Rad inhibition display fundamental differences in their sensitivity to PKA regulation. A caveat here is we cannot discount a contribution of Ca_V1.2 channels which are not bound to Rad[R208A/L235A] to the observed forskolin-induced increase in I_Ba. However, the finding that Rad[R208A/L235A] inhibits cardiac Ca_V1.2 to almost the same extent as WT Rad (Fig. 4 E and F) suggests Rad[R208A/L235A]-bound channels predominate over unbound channels, and is consistent with the interpretation that Ca_V1.2 channels undergoing BBI Rad inhibition are up-regulated by PKA activation.

Fig. 4.

Rad[R208A/L235A] does not bind Ca_Vβ and selectively inhibits Ca_V1.2 and Ca_V2.2 channels. (A) Crystal structure of Rad G domain with residues R208 and L235 highlighted. (B) FRET efficiency measurements in HEK293 cells coexpressing CFP-FRB + β₃-YFP (control), CFP-Rad + β₃-YFP, or CFP-Rad[R208A/L235A] + β₃-YFP. Data are means ± SEM. *P < 0.01, one-way ANOVA. (C) Binding-curve analyses of FRET experiments. (D) Population I_peak–V plots for cells expressing α_1C + β_2a (black squares; n = 11), α_1C + β_2a + Rad[R208A/L235A] (red squares; n = 10); α_1D + β_2a (black circles; n = 10), α_1D + β_2a + Rad[R208A/L235A] (red circles; n = 13); α_1A + β_2a (black triangles; n = 10), α_1A + β_2a + Rad[R208A/L235A] (red triangles; n = 11); and α_1B + β_2a (black diamonds; n = 10), α_1B + β_2a + Rad[R208A/L235A] (red diamonds; n = 14). P < 0.05, Student’s t test. (E) Exemplar Ba²⁺ currents from cultured adult guinea pig ventricular cardiomyocytes expressing Rad (Top) and Rad[R208A/L235A] (Bottom). (F) Population I_peak–V for cardiomyocytes expressing either CFP-Rad (red squares; n = 4) or CFP-Rad[R208A/L235A] (blue squares; n = 8). Dotted line is mean current density for control cardiomyocytes expressing GFP, reproduced from Fig. 2G. (G) Exemplar currents (Top) and diary plot (Bottom) showing the impact of 1 μM forskolin on I_Ba in cardiomyocytes expressing CFP-Rad[R208A/L235A]. (H) Bar chart showing differential impact of forskolin in up-regulating Ca_V1.2 I_Ba in cardiomyocytes expressing CFP-Rad or CFP-Rad[R208A/L235A]. Data are means ± SEM.

Rem[R200A/L227A] and Rad[R208A/L235A] Inhibit HVA Ca_V Channels in Dorsal Root Ganglion Neurons.

Finally, we determined the performance of Rem[R200A/L227A] and Rad[R208A/L235A] as Ca_V channel inhibitors in primary cells with a complex expression of multiple Ca_V channel types. We chose dorsal root ganglion neurons which express multiple HVA Ca_V1/Ca_V2 channels as well as low voltage-activated (LVA) Ca_V3.2 channels. Mouse DRG neurons express mostly Ca_V2.1 and Ca_V2.2, with a smaller contribution of Ca_V1.2 and Ca_V2.3 channels (21). We used adenoviral vectors to robustly express GFP (control) or CFP-tagged RGKs in cultured mouse DRG neurons (Fig. 5A). In control cells, a ramp protocol elicited two components of I_Ba, reflecting currents through LVA and HVA Ca_V channels, respectively. Overexpressing WT Rad essentially eliminated the HVA current component while leaving the LVA element intact (Fig. 5B). We further assessed the impact of various WT and mutant RGKs on the HVA Ca_V channel currents using step depolarizations. For these experiments, LVA Ca_V channel currents were eliminated by 5 μM mibefradil and a −50-mV holding potential. Control DRG neurons displayed HVA I_Ba currents which were dramatically reduced by WT Rad (Fig. 5 C and D; I_peak,−10mV = −76.5 ± 13.8 pA/pF, n = 10 for GFP compared with I_peak,−10mV = −3.5 ± 1.3 pA/pF, n = 19 for CFP-Rad; SI Appendix, Fig. S6). DRG neurons expressing CFP-Rad[R208A/L235A] showed a significant 62% decrease in HVA I_Ba compared with control (Fig. 5 C and D; I_peak,−10mV = −29.8 ± 5.8 pA/pF, n = 13; SI Appendix, Fig. S6). Similar to WT Rad, DRG neurons expressing either CFP-Rem or CFP-Gem showed a dramatically reduced HVA I_Ba amplitude (Fig. 5D; I_peak,−10mV = −8.7 ± 3.8 pA/pF, n = 8 for CFP-Rem, and I_peak,−10mV = −4.8 ± 0.9 pA/pF, n = 4 for CFP-Gem; SI Appendix, Fig. S6). Expressing CFP-Rem[R200A/L227A] depressed HVA I_Ba by 25% compared with control (Fig. 5D; I_peak,−10mV = −56.5 ± 6.6 pA/pF, n = 13; SI Appendix, Fig. S6), substantially less than the reduction observed with CFP-Rad[R208A/L235A]. By contrast, Gem[R196A/V223A] had no impact on HVA I_Ba in DRG neurons (Fig. 5D; I_peak,−10mV = −74.5 ± 15.4 pA/pF, n = 12; SI Appendix, Fig. S6). Overall, the rank order of inhibition of HVA I_Ba by these mutant RGKs is consistent with the notion that CFP-Rad[R208A/L235A] inhibits both Ca_V1.2 and Ca_V2.2, CFP-Rem[R200A/L227A] inhibits only Ca_V1.2, and Gem[R196A/V223A] is inert against HVA Ca_V channels.

Fig. 5.

Differential block of high voltage-activated Ca_V channel currents in DRG neurons by WT and mutant RGK proteins. (A) Representative images of cultured DRG neurons expressing GFP, CFP-Rad, or CFP-Rad[R208A/L235A]. (B) Exemplar I_Ba waveforms elicited by voltage-ramp protocols in DRG neurons expressing GFP (Left) or CFP-Rad (Right). (C) Exemplar family of HVA I_Ba from DRG neurons expressing GFP (Left), CFP-Rad (Middle), or CFP-Rad[208A/L235A] (Right). Currents were elicited from a holding potential of −50 mV and in the presence of 1 μM mibefradil to eliminate LVA T-type currents. (D) Bar chart showing the relative impact of distinct WT and β-binding–deficient mutant RGKs on HVA Ca_V1/Ca_V2 channel currents in cultured DRG neurons. Data are means ± SEM. *P < 0.05, one-way ANOVA and post hoc Bonferroni test.

Discussion

Pharmacological blockade of distinct Ca_V1/Ca_V2 channel types is an important actual or potential therapy for many diseases, including hypertension (Ca_V1.2), angina (Ca_V1.2), cardiac arrhythmias (Ca_V1.2), chronic pain (Ca_V2.2), stroke (Ca_V2), and Parkinson’s disease (Ca_V1.3) (1, 22, 23). Ca_V1 channels are effectively blocked by dihydropyridines, benzothiazepines, and phenylalkylamines, while Ca_V2 channels are inhibited by various animal venoms: ω-agatoxin IVA (Ca_V2.1), ω-conotoxins GVIA and MVIIA (Ca_V2.2), and SNX-482 (Ca_V2.3) (24). Prialt (ziconotide), a blocker of Ca_V2.2 derived from a marine snail conotoxin, is Food and Drug Administration-approved for the treatment of chronic pain (25). The use of small-molecule Ca_V1/Ca_V2 channel blockers is mainly limited by two factors. First, Ca_V1/Ca_V2 expression in many types of excitable cells risks prohibitive off-target effects. Second, due to a high degree of similarity among pore-forming α₁-subunits (e.g., the L-type channels, Ca_V1.1 to Ca_V1.4), currently available small-molecule blockers may not effectively distinguish between Ca_V channels of the same class. Difficulties encountered in developing Ca_V1.3-selective blockers as a potential treatment for Parkinson’s disease exemplify these challenges (26, 27). Efficacy of such a treatment approach was suggested by reports that the reliance of substantia nigra neurons on Ca_V1.3 for pacemaking made them sensitive to Ca²⁺ overload and vulnerable to cell death which drives the development of Parkinson’s disease (28, 29). Epidemiological studies suggest indeed some beneficial effects of L-type calcium channel (LTCC) blockers in Parkinson’s disease (30). However, because the currently available LTCC blockers are not selective for Ca_V1.3, off-target effects (e.g., on cardiovascular Ca_V1.2 channels) risk serious side effects such as hypotension, significantly narrowing the therapeutic window (31).

Genetically encoded Ca_V channel blockers could offer an alternative solution without the above-mentioned drawbacks of small-molecule inhibitors. Off-target effects might be avoided by restricted expression in target tissues or defined cell populations (9, 10). RGKs are promising candidates for such an alternative treatment approach, given their potency as Ca_V channel blockers. Their potential usefulness is twofold: (i) as endogenous GECCIs for therapeutic or biotechnological applications, and (ii) as natural prototypes that can help inform strategies to design novel GECCIs for targeted applications in diseases involving excitable cells. Regarding the former, the indiscriminate nature of RGK inhibition of all Ca_V1/Ca_V2 channels represents a potential obstacle for some applications. We tested here whether selectivity for particular Ca_V1/Ca_V2 isoforms could be engineered into RGKs. Based on the intuition that the indiscriminate manner with which RGKs inhibit all Ca_V1/Ca_V2 channel types is a consequence of their binding to auxiliary Ca_Vβ-subunits, we mutated RGKs to eliminate their capacity of binding to Ca_Vβ. This simple maneuver revealed Rem[R200A/L227A] as a Ca_V1.2-selective blocker and Rad[R208A/L235A] as a selective inhibitor for Ca_V1.2/Ca_V2.2. The selectivity of Rem[R200A/L227A] for Ca_V1.2 over Ca_V1.3 is noteworthy, given that currently available small-molecule LTCC blockers do not distinguish these channels. Hence, Rem[R200A/L227A] could be a valuable tool for differentially blocking Ca_V1.2- and Ca_V1.3-mediated signaling in excitable cells, such as many types of neurons, that coexpress both channel types. Similarly, Rad[R208A/L235A] could be applied to examine Ca_V1.2/Ca_V2.2-dependent signaling pathways. The effectiveness of both Rem[R200A/L227A] and Rad[R208A/L235A] in blocking HVA Ca_V channels in heart cells and DRG neurons demonstrates their utility as selective GECCIs. Additionally, our experiments revealed the existence of BBI mechanisms underlying Rem and Rad inhibition of Ca_V1/Ca_V2 channels in excitable cells. This raises the question of the biological significance of BBD versus BBI Ca_V channel inhibition by RGKs. Our findings in cardiac myocytes suggest indeed functionally relevant differences between the two inhibitory mechanisms. Cardiac Ca_V1.2 channels are acutely up-regulated pharmacologically by agonists such as BAY K 8644 or physiological activation of PKA initiated by β-adrenergic agonists. The latter contributes to the fight-or-flight response. Rem-inhibited Ca_V1.2 channels in heart cells can be overridden by BAY K 8644, indicating that the blocked channels remain at the cell surface (32). However, both WT Rem- and Rad-inhibited channels are insensitive to PKA-mediated regulation (20, 32). By contrast, we found that Ca_V1.2 channels inhibited by either Rem[R200A/L227A] or Rad[R208A/L235A] can be robustly up-regulated by PKA. These results suggest that cardiac Ca_V1.2 channels inhibited by Rem or Rad through the BBD mechanism are electrically silent, while those inhibited by the BBI pathway are coincidentally activated by membrane depolarization and PKA-mediated phosphorylation. This paradigm could solve the conundrum of how a subset of Ca_V1.2 channels in heart cells might be reserved for signaling functions other than contraction, given that these channels are voltage-gated and the cardiac sarcolemma is subject to excitation with each heartbeat (33, 34). In this regard, it is noteworthy that GDP-bound Rem and Rad have a lower affinity for Ca_Vβ than their GTP-loaded counterparts (35). We speculate that endogenous Rad toggles between BBD and BBI mechanisms to inhibit cardiac Ca_V1.2 channels dependent on the G domain being bound to GTP or GDP. Testing this proposition will be an interesting concept for future experiments.

Materials and Methods

Detailed methods are provided in SI Appendix, Materials and Methods.

Cell Culture and Transfection.

Low‐passage‐number HEK293 cells were transiently transfected with Ca_Vα (6 μg), Ca_Vβ (4 μg), T antigen (2 μg), and RGKs (4 μg) using the calcium‐phosphate precipitation method.

Primary Cell Isolation and Culture.

Primary cultures of adult guinea pig heart cells and murine DRG neurons were prepared and infected with adenovirus as described (14, 36, 37). Procedures were in accordance with the guidelines of the Columbia University Animal Care and Use Committee.

Molecular Biology, Plasmids, and Adenoviral Vectors.

Generation of XFP-tagged RGKs (Rad, Rem, Rem2, and Gem) and β_2a,TM has been previously described (12, 15). Adenoviral vectors were generated using the AdEasy XL System (Stratagene) as previously described (38).

Electrophysiology.

Whole‐cell recordings were carried out at room temperature on HEK293 cells 48 to 72 h after transfection as previously described (12). Whole-cell currents were recorded from isolated ventricular myocytes as described (36, 39).

Fluorescence Resonance Energy Transfer Imaging.

We used three‐cube FRET to probe protein–protein interactions (35). Relative K_d and E_max values were calculated as previously described (35, 40).

Acknowledgments

We thank Ming Chen for excellent technical support, and Dr. Shingo Kariya (Columbia University) for help with isolation of DRG neurons. This work was supported by Grants R01 HL 084332 and 1R01-GM107585 from the National Institutes of Health (to H.M.C.). A.A.P. was supported by an AHA predoctoral fellowship (13PRE13970018) and a UNCF-Merck graduate dissertation fellowship (CU11-2479). Z.S. was supported by an institutional training grant (T32HL120826). J.S. is now an employee and stock owner of Biogen; this work was completed before he joined the company. Biogen did not have a role in the design, conduct, analysis, interpretation, or funding of the research related to this work.

Footnotes

↵¹A.A.P., D.D.C., and Z.S. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: hc2405{at}cumc.columbia.edu.

Author contributions: A.A.P., D.D.C., and H.M.C. designed research; A.A.P., D.D.C., Z.S., P.C., and H.M.C. performed research; J.S. contributed new reagents/analytic tools; A.A.P., D.D.C., Z.S., P.C., and H.M.C. analyzed data; and A.A.P., J.S., and H.M.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1811024115/-/DCSupplemental.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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(2012) Ca_V1.3-selective L-type calcium channel antagonists as potential new therapeutics for Parkinson’s disease. Nat Commun 3:1146.
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1. Huang H, et al.
(2014) Modest Ca_V1.342-selective inhibition by compound 8 is β-subunit dependent. Nat Commun 5:4481.
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↵
1. Surmeier DJ,
2. Schumacker PT
(2013) Calcium, bioenergetics, and neuronal vulnerability in Parkinson’s disease. J Biol Chem 288:10736–10741.
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↵
1. Guzman JN, et al.
(2010) Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468:696–700.
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1. Ritz B, et al.
(2010) L-type calcium channel blockers and Parkinson disease in Denmark. Ann Neurol 67:600–606.
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1. Simuni T, et al.
(2010) Tolerability of isradipine in early Parkinson’s disease: A pilot dose escalation study. Mov Disord 25:2863–2866.
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↵
1. Xu X,
2. Marx SO,
3. Colecraft HM
(2010) Molecular mechanisms, and selective pharmacological rescue, of Rem-inhibited Ca_V1.2 channels in heart. Circ Res 107:620–630.
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↵
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2. Colecraft HM
(2013) L-type calcium channel targeting and local signalling in cardiac myocytes. Cardiovasc Res 98:177–186.
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(2006) Dichotomy of Ca²⁺ in the heart: Contraction versus intracellular signaling. J Clin Invest 116:623–626.
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2. Colecraft HM
(2015) Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation. J Physiol 593:5075–5090.
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Proceedings of the National Academy of Sciences: 115 (47)

Table of Contents

Submit

[1] ↵
Zamponi GW,
Striessnig J,
Koschak A,
Dolphin AC
(2015) The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential. Pharmacol Rev 67:821–870.
OpenUrl Abstract/FREE Full Text

[2] Zamponi GW,

[3] Striessnig J,

[4] Koschak A,

[5] Dolphin AC

[6] ↵
Buraei Z,
Yang J
(2010) The β subunit of voltage-gated Ca²⁺ channels. Physiol Rev 90:1461–1506.
OpenUrl CrossRef PubMed

[7] Buraei Z,

[8] Yang J

[9] ↵
Dolphin AC
(2016) Voltage-gated calcium channels and their auxiliary subunits: Physiology and pathophysiology and pharmacology. J Physiol 594:5369–5390.
OpenUrl CrossRef PubMed

[10] Dolphin AC

[11] ↵
Ben-Johny M,
Yue DT
(2014) Calmodulin regulation (calmodulation) of voltage-gated calcium channels. J Gen Physiol 143:679–692.
OpenUrl Abstract/FREE Full Text

[12] Ben-Johny M,

[13] Yue DT

[14] ↵
Adams PJ,
Ben-Johny M,
Dick IE,
Inoue T,
Yue DT
(2014) Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation. Cell 159:608–622.
OpenUrl CrossRef PubMed

[15] Adams PJ,

[16] Ben-Johny M,

[17] Dick IE,

[18] Inoue T,

[19] Yue DT

[20] ↵
Béguin P, et al.
(2001) Regulation of Ca²⁺ channel expression at the cell surface by the small G-protein kir/Gem. Nature 411:701–706.
OpenUrl CrossRef PubMed

[21] Béguin P, et al.

[22] ↵
Yang T,
Colecraft HM
(2013) Regulation of voltage-dependent calcium channels by RGK proteins. Biochim Biophys Acta 1828:1644–1654.
OpenUrl CrossRef PubMed

[23] Yang T,

[24] Colecraft HM

[25] ↵
Finlin BS,
Crump SM,
Satin J,
Andres DA
(2003) Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases. Proc Natl Acad Sci USA 100:14469–14474.
OpenUrl Abstract/FREE Full Text

[26] Finlin BS,

[27] Crump SM,

[28] Satin J,

[29] Andres DA

[30] ↵
Xu X,
Colecraft HM
(2009) Engineering proteins for custom inhibition of Ca(V) channels. Physiology (Bethesda) 24:210–218.
OpenUrl CrossRef PubMed

[31] Xu X,

[32] Colecraft HM

[33] ↵
Murata M,
Cingolani E,
McDonald AD,
Donahue JK,
Marbán E
(2004) Creation of a genetic calcium channel blocker by targeted gem gene transfer in the heart. Circ Res 95:398–405.
OpenUrl Abstract/FREE Full Text

[34] Murata M,

[35] Cingolani E,

[36] McDonald AD,

[37] Donahue JK,

[38] Marbán E

[39] ↵
Makarewich CA, et al.
(2012) A caveolae-targeted L-type Ca²⁺ channel antagonist inhibits hypertrophic signaling without reducing cardiac contractility. Circ Res 110:669–674.
OpenUrl Abstract/FREE Full Text

[40] Makarewich CA, et al.

[41] ↵
Yang T,
Xu X,
Kernan T,
Wu V,
Colecraft HM
(2010) Rem, a member of the RGK GTPases, inhibits recombinant Ca_V1.2 channels using multiple mechanisms that require distinct conformations of the GTPase. J Physiol 588:1665–1681.
OpenUrl CrossRef PubMed

[42] Yang T,

[43] Xu X,

[44] Kernan T,

[45] Wu V,

[46] Colecraft HM

[47] ↵
Puckerin AA,
Chang DD,
Subramanyam P,
Colecraft HM
(2016) Similar molecular determinants on Rem mediate two distinct modes of inhibition of Ca_V1.2 channels. Channels (Austin) 10:379–394.
OpenUrl

[48] Puckerin AA,

[49] Chang DD,

[50] Subramanyam P,

[51] Colecraft HM

[52] ↵
Yang T,
He LL,
Chen M,
Fang K,
Colecraft HM
(2013) Bio-inspired voltage-dependent calcium channel blockers. Nat Commun 4:2540.
OpenUrl PubMed

[53] Yang T,

[54] He LL,

[55] Chen M,

[56] Fang K,

[57] Colecraft HM

[58] ↵
Yang T,
Puckerin A,
Colecraft HM
(2012) Distinct RGK GTPases differentially use α1- and auxiliary β-binding-dependent mechanisms to inhibit Ca_V1.2/Ca_V2.2 channels. PLoS One 7:e37079.
OpenUrl CrossRef PubMed

[59] Yang T,

[60] Puckerin A,

[61] Colecraft HM

[62] ↵
Béguin P, et al.
(2007) RGK small GTP-binding proteins interact with the nucleotide kinase domain of Ca²⁺-channel beta-subunits via an uncommon effector binding domain. J Biol Chem 282:11509–11520.
OpenUrl Abstract/FREE Full Text

[63] Béguin P, et al.

[64] ↵
Beqollari D,
Romberg CF,
Filipova D,
Papadopoulos S,
Bannister RA
(2015) Functional assessment of three Rem residues identified as critical for interactions with Ca(2+) channel β subunits. Pflugers Arch 467:2299–2306.
OpenUrl CrossRef

[65] Beqollari D,

[66] Romberg CF,

[67] Filipova D,

[68] Papadopoulos S,

[69] Bannister RA

[70] ↵
Xu X,
Zhang F,
Zamponi GW,
Horne WA
(2015) Solution NMR and calorimetric analysis of Rem2 binding to the Ca²⁺ channel β4 subunit: A low affinity interaction is required for inhibition of Cav2.1 Ca²⁺ currents. FASEB J 29:1794–1804.
OpenUrl CrossRef PubMed

[71] Xu X,

[72] Zhang F,

[73] Zamponi GW,

[74] Horne WA

[75] ↵
Fan M,
Buraei Z,
Luo HR,
Levenson-Palmer R,
Yang J
(2010) Direct inhibition of P/Q-type voltage-gated Ca²⁺ channels by Gem does not require a direct Gem/Ca_vbeta interaction. Proc Natl Acad Sci USA 107:14887–14892.
OpenUrl Abstract/FREE Full Text

[76] Fan M,

[77] Buraei Z,

[78] Luo HR,

[79] Levenson-Palmer R,

[80] Yang J

[81] ↵
Wang G, et al.
(2010) Rad as a novel regulator of excitation-contraction coupling and beta-adrenergic signaling in heart. Circ Res 106:317–327.
OpenUrl Abstract/FREE Full Text

[82] Wang G, et al.

[83] ↵
Murali SS, et al.
(2015) High-voltage-activated calcium current subtypes in mouse DRG neurons adapt in a subpopulation-specific manner after nerve injury. J Neurophysiol 113:1511–1519.
OpenUrl CrossRef PubMed

[84] Murali SS, et al.

[85] ↵
Triggle DJ
(2007) Calcium channel antagonists: Clinical uses—Past, present and future. Biochem Pharmacol 74:1–9.
OpenUrl CrossRef PubMed

[86] Triggle DJ

[87] ↵
Valentino K, et al.
(1993) A selective N-type calcium channel antagonist protects against neuronal loss after global cerebral ischemia. Proc Natl Acad Sci USA 90:7894–7897.
OpenUrl Abstract/FREE Full Text

[88] Valentino K, et al.

[89] ↵
Simms BA,
Zamponi GW
(2014) Neuronal voltage-gated calcium channels: Structure, function, and dysfunction. Neuron 82:24–45.
OpenUrl CrossRef PubMed

[90] Simms BA,

[91] Zamponi GW

[92] ↵
Wermeling DP
(2005) Ziconotide, an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain. Pharmacotherapy 25:1084–1094.
OpenUrl CrossRef PubMed

[93] Wermeling DP

[94] ↵
Kang S, et al.
(2012) Ca_V1.3-selective L-type calcium channel antagonists as potential new therapeutics for Parkinson’s disease. Nat Commun 3:1146.
OpenUrl CrossRef PubMed

[95] Kang S, et al.

[96] ↵
Huang H, et al.
(2014) Modest Ca_V1.342-selective inhibition by compound 8 is β-subunit dependent. Nat Commun 5:4481.
OpenUrl CrossRef PubMed

[97] Huang H, et al.

[98] ↵
Surmeier DJ,
Schumacker PT
(2013) Calcium, bioenergetics, and neuronal vulnerability in Parkinson’s disease. J Biol Chem 288:10736–10741.
OpenUrl Abstract/FREE Full Text

[99] Surmeier DJ,

[100] Schumacker PT

[101] ↵
Guzman JN, et al.
(2010) Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468:696–700.
OpenUrl CrossRef PubMed

[102] Guzman JN, et al.

[103] ↵
Ritz B, et al.
(2010) L-type calcium channel blockers and Parkinson disease in Denmark. Ann Neurol 67:600–606.
OpenUrl CrossRef PubMed

[104] Ritz B, et al.

[105] ↵
Simuni T, et al.
(2010) Tolerability of isradipine in early Parkinson’s disease: A pilot dose escalation study. Mov Disord 25:2863–2866.
OpenUrl CrossRef PubMed

[106] Simuni T, et al.

[107] ↵
Xu X,
Marx SO,
Colecraft HM
(2010) Molecular mechanisms, and selective pharmacological rescue, of Rem-inhibited Ca_V1.2 channels in heart. Circ Res 107:620–630.
OpenUrl Abstract/FREE Full Text

[108] Xu X,

[109] Marx SO,

[110] Colecraft HM

[111] ↵
Shaw RM,
Colecraft HM
(2013) L-type calcium channel targeting and local signalling in cardiac myocytes. Cardiovasc Res 98:177–186.
OpenUrl CrossRef PubMed

[112] Shaw RM,

[113] Colecraft HM

[114] ↵
Molkentin JD
(2006) Dichotomy of Ca²⁺ in the heart: Contraction versus intracellular signaling. J Clin Invest 116:623–626.
OpenUrl CrossRef PubMed

[115] Molkentin JD

[116] ↵
Chang DD,
Colecraft HM
(2015) Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation. J Physiol 593:5075–5090.
OpenUrl

[117] Chang DD,

[118] Colecraft HM

[119] ↵
Miriyala J,
Nguyen T,
Yue DT,
Colecraft HM
(2008) Role of Ca_Vbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac Ca_V1.2 channels. Circ Res 102:e54–e64.
OpenUrl Abstract/FREE Full Text

[120] Miriyala J,

[121] Nguyen T,

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