Rad protein: An essential player in L-type Ca²⁺ channel localization and modulation in cardiomyocytes

L-type Ca²⁺ channels (LTCCs, cardiac-specific subtype Ca_v1.2) are the primary gateway for Ca²⁺ entry (I_Ca,L) in cardiomyocytes and are essential for excitation–contraction (EC) coupling. In adult ventricular cardiomyocytes, I_Ca,L predominantly localizes to transverse (t-) tubules where they lie in close apposition to junctional sarcoplasmic reticulum (SR). During EC coupling, I_Ca,L triggers SR Ca²⁺ release, which initiates contraction.

Ca_v1.2 is a key point of (dys)regulation in health and disease, for example, changes in Ca_v1.2 activity, sarcolemmal density, or subcellular organization have been shown to result in cardiac arrhythmias, heart failure and sudden cardiac death (Best and Kamp, 2012). Ca_v1.2 is a multi-subunit protein comprised of a pore-forming α_1C and regulatory β_2B and α_2δ subunits. During the fight-or-flight response, β-adrenergic signaling stimulates I_Ca,L. A long-standing view of the underlying mechanism has involved protein kinase A (PKA)-dependent phosphorylation of Ca_v1.2 α_1c and/or β_2B subunits, however, studies carried out over the past two decades have challenged this view (compare reviews Kamp and Hell, 2000; Benitah et al., 2010; Colecraft, 2020). Indeed, amino acid truncation and/or substitution studies have suggested that the proposed regulatory residues on α_1c and β_2B are dispensable for β-adrenergic activation of I_Ca,L.

Ras associated with diabetes (Rad) (Reynet and Kahn, 1993) belongs to the highly conserved RGK subfamily of small GTP-binding proteins (Rad, Rem, Rem2, and Gem/Kir, see Fig. 1, inset) that regulate LTCCs (Colecraft, 2020). Within this subfamily, Rad (Reynet and Kahn, 1993) and Rem (Finlin and Andres, 1997) are predominantly expressed in the heart. Although the Ras superfamily is typically associated with cell growth, proliferation, and adaptation (Wennerberg et al., 2005), Rad has emerged as a major LTCC modulator. It is suggested that Rad inhibits I_Ca,L via an association with the β₂ auxiliary subunit (e.g., Finlin et al., 2003) and that during β-adrenergic signaling, this inhibition is reduced (e.g., (Liu et al, 2020). Clarification of how Rad regulates LTCC and cardiomyocyte function may become more important in the disease state, where Ca²⁺ dysregulation is a critical issue.

Investigating the effects of endogenous Rad on LTCC is challenging. Early studies carried out in heterologous expression systems showed that Rad and other RGK subfamily members can suppress LTCC activity (Béguin et al, 2001; Finlin et al, 2003, 2006; Ward et al, 2004). From this work, it was initially proposed (Béguin et al, 2001) that the underlying mechanism could involve channel trafficking, however, later studies showed that Rad can modulate channel activity directly and that this interaction requires the presence of β_2B, at least in these models (e.g., Finlin et al., 2003). To overcome challenges in recapitulating native organization and stoichiometry within the LTCC multimeric complex in these expression models, Wang et al. (2010) used silencing RNA in cultured adult rat cardiomyocytes to demonstrate an inhibitory effect of endogenous Rad. While this enabled the investigation of endogenous Rad function, the method involved short-term cell culture which is associated with cardiomyocyte dedifferentiation. Nevertheless, subsequent development of a global (Manning et al., 2013), then cardiac-specific (Ahern et al, 2019) Rad knock-out mouse models have further supported a role for Rad in regulating I_Ca,L. In an earlier issue of JGP, Elmore et al. (2024) investigated the role of the Rad C-terminus by generating full-length and C-terminally truncated Rad knock-in mice. By attaching an N-terminal 3xFlag affinity tag, they could also visualize wild-type and truncated Rad protein distribution in isolated adult ventricular cardiomyocytes.

Studies in expression systems, cultured, and freshly isolated cardiomyocytes show that reduced Rad expression is associated with increased I_Ca,L density, leftward shift of the voltage-dependence of activation and little change to the steady-state voltage-dependent inactivation (Finlin et al, 2003; Wang et al, 2010; Manning et al, 2013; Ahern et al, 2019). A recent study by Liu et al. (2020) using proximity proteomics showed that Rad is located in close proximity to α_1C and β_2B in ventricular cardiomyocytes. In the earlier issue, Elmore et al. (2024) used anti-FLAG immunocytochemistry to show a striated distribution of Rad that has a similar visual registration as that of α_1C labelling. Certainly, further investigation into Rad distribution and its role in I_Ca,L regulation in cardiomyocytes from atria (such as in rat) and from larger mammals that have lower t-tubular density remains to be explored.

Though it has been shown that inhibition of I_Ca,L by Rad requires β_2B (e.g., Finlin et al., 2003), the details of this mechanism remain unclear. Studies with Rem have shown that its inhibition of I_Ca,L depends on its C-terminus, which is (also) responsible for its localization to the plasma membrane and interaction with phosphatidylinositol (PIP) lipids (Correll et al., 2007; Xu et al., 2010). Elmore et al. (2024) tested this idea for Rad by introducing a stop codon at Ala277. They showed that truncated Rad abolished the inhibitory effect of full-length Rad on I_Ca,L (Fig. 1), as well as its striated distribution. Elmore et al. (2024) go on to carry out further analysis using AlphaFold, which predicted a membrane interacting domain at helix 8 of Rad, which was deleted in truncated Rad. Elmore et al. (2024) observed that labelling of truncated Rad did not show a striated pattern and this may indicate that the deletion may have also disrupted the Rad–β_2B interaction. If Rad links Ca_v1.2, PKA, and PIP, it might also interact with other scaffolding proteins including A kinase anchoring proteins (AKAPs), caveolin-3, and/or lipid rafts. Although previous studies have shown that PKA-dependent stimulation of I_Ca,L is partly dependent on caveolin-3 (e.g., Bryant et al., 2018), fusion of a caveolin-3 binding domain to Rem did not alter basal I_Ca,L or its response to PKA in adult feline cardiomyocytes (Correll et al., 2017). Nevertheless, this possible connection, alongside a possible involvement of AKAPs and the cytoskeleton (e.g., Bilan et al., 1998; Ward et al., 2002) may also suggest a role for Rad in the maintenance of microdomains, t-tubules, or other structures.

A key aspect to the interaction between Rad and Ca_v1.2 is that it provides the basis for the effects of PKA on I_Ca,L. Previous studies have shown that a reduction in Rad expression was associated with a reduction (Wang et al., 2010) or loss (Manning et al, 2013; Ahern et al, 2019) of isoprenaline’s stimulatory effect on I_Ca,L. On the other hand, while overexpression of Rad reduced I_Ca,L, this effect was not relieved with isoprenaline (Wang et al., 2010; see also Xu et al., 2010 for Rem). More recently, Liu et al. (2020) showed that the close association of Rad with α_1C and β_2B is reduced with isoprenaline treatment. They also showed that mutation of 35 proposed PKA phosphorylation sites on α_1C and 28 on β_2B did not impair the isoprenaline effect, supporting previous studies that found phosphorylation of Ca_v1.2 to be unnecessary for PKA stimulation. Instead, the mechanism appears to require 4 serine residues on Rad (Papa et al, 2022). The data obtained by Elmore et al. (2024) lends further support to a role for endogenous Rad in PKA modulation of I_Ca,L. In the earlier issue, they showed that isoprenaline treatment was associated with reduced t-tubular 3xFLAG-Rad labelling (Fig. 1). Moreover, isoprenaline was not effective in modulating I_Ca,L in cells from the truncated Rad mice. Thus, these studies suggest that phosphorylation sites on Ca_v1.2 may at best modulate the inhibitory effect of Rad though any role remains to be clarified.

Alterations in Rad expression have also been associated with other changes in cellular Ca²⁺ handling. Several studies have reported that reduced Rad expression is associated with larger Ca²⁺ transients (Wang et al, 2010; Manning et al, 2013; Ahern et al, 2019), without an effect on the Na⁺–Ca²⁺ exchanger (Wang et al., 2010). An effect on SR Ca²⁺ content (Wang et al., 2010; Manning et al., 2013 versus Ahern et al, 2019), or diastolic Ca²⁺ movements (Wang et al., 2010 versus Manning et al., 2013) remain to be clarified. While it is unclear whether Rad has other targets, heart tissue from the global Rad knock-out (KO) mouse showed increased phosphorylation of both calmodulin-dependent kinase 2 and phospholamban (Manning et al., 2013). Moreover, the loss of the frequency-dependence of diastolic and systolic Ca²⁺ levels associated with Rad KO was recovered with KN-93 treatment (Manning et al., 2013). While these changes have not been reported in cardiac-specific KO or Rad truncation mouse models, they highlight the possibility that Rad could be a point of regulatory crosstalk and local organization. Indeed, RGK proteins contain interaction domains for calmodulin and 14-3-3 proteins (Kelly, 2005).

As Elmore et al. (2024) discuss, the Rad–β_2B interaction is a potential therapeutic target. Chang et al. (2007) reported a reduction in Rad expression in human heart failure, while a single nucleotide polymorphism has been identified in the Rad gene in patients with congestive heart failure (Lynch et al., 2002). Elmore et al. (2024) showed that truncation of the Rad C-terminus increased left ventricular ejection fraction and abolished a β-adrenergic response. Meanwhile, Manning et al. (2013) observed increased spontaneous Ca²⁺ and electrical activity in cardiomyocytes from the global Rad KO mouse. Thus, fine tuning of I_Ca,L remains an important challenge to maintain contractile capacity whilst avoiding arrhythmogenic behavior and Ca²⁺-dependent cytotoxicity.

Elmore et al. (2024) suggest that a possible route to targeting the Rad–β_2B interaction could be PIP. Although therapeutic PIP has some precedence (Burg et al., 2022; Murata et al., 2004), PIP is involved in many cellular processes including t-tubule formation, membrane trafficking and maintaining electrostatic effects. Further work clarifying how PIP interacts with Rad or RGK proteins will be useful in guiding these strategies, as well as exploring novel approaches to tune Rad proteins (Xie et al., 2023). Similarly, LTCC subpopulations (Best and Kamp, 2012) should be considered in this aspect.

In summary, the intricate interplay between Rad and Ca_v1.2 underscores the complexity of cellular signaling mechanisms and the need for meticulous experimental design to gain more insights into the underlying molecular mechanisms. With the elegant use of transgenic mouse models, Elmore et al. (2024) provide new insights into Rad-dependent LTCC modulation. The importance of Rad structure in LTCC modulation adds novelty in the growing field of designing new therapeutic targets.

David A. Eisner served as editor.

Work in the authors’ laboratories is supported by funding provided by KU Leuven (grant STG/23/044, E. Dries) and British Heart Foundation (grant FS/IBSRF/24/25203, C.H.T. Kong).