Robert N. Correll

A Caveolae-Targeted L-Type Ca2+ Channel Antagonist Inhibits Hypertrophic Signaling Without Reducing Cardiac Contractility

Rationale: The source of Ca2+ to activate pathological cardiac hypertrophy is not clearly defined. Ca2+ influx through the L-type Ca2+ channels (LTCCs) determines “contractile” Ca2+, which is not thought to be the source of “hypertrophic” Ca2+. However, some LTCCs are housed in caveolin-3 (Cav-3)–enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown. Objective: To test the idea that LTCCs in Cav-3–containing signaling domains are a source of Ca2+ to activate the calcineurin–nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy. Methods and Results: We developed reagents that targeted Ca2+ channel-blocking Rem proteins to Cav-3–containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca2+ influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility. Conclusions: We provide proof of concept that Ca2+ influx through LTCCs within caveolae signaling domains can activate “hypertrophic” signaling, and this Ca2+ influx can be selectively blocked without reducing cardiac contractility.

Regulation of L-type Ca2+ Channel Activity and Insulin Secretion by the Rem2 GTPase

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions and are controlled by a diversity of intracellular signals. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. In this study, microarray analysis of the mouse insulinoma MIN6 cell line revealed that the transcription of Rem2 gene is strongly induced by exposure to high glucose, which was confirmed by real-time reverse transcriptase-PCR and RNase protection analysis. Because elevation of intracellular Ca2+ in pancreatic β-cells is essential for insulin secretion, we tested the hypothesis that Rem2 attenuates Ca2+ currents to regulate insulin secretion. Co-expression of Rem2 with CaV 1.2 or CaV1.3 L-type Ca + channels in a heterologous expression system completely inhibits de novo Ca2+ current expression. In addition, ectopic overexpression of Rem2 both inhibited L-type Ca2+ channel activity and prevented glucose-stimulated insulin secretion in pancreatic β-cell lines. Co-immunoprecipitation studies demonstrate that Rem2 associates with a variety of CaVβ subunits. Importantly, surface biotinylation studies demonstrate that the membrane distribution of Ca2+ channels was not reduced at a time when channel activity was potently inhibited by Rem2 expression, indicating that Rem2 modulates channel function without interfering with membrane trafficking. Taken together, these data suggest that inhibition of L-type Ca2+ channels by Rem2 signaling may represent a new and potentially important mechanism for regulating Ca2+-triggered exocytosis in hormone-secreting cells, including insulin secretion in pancreatic β-cells.

Analysis of the Complex between Ca2+ Channel β-Subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary β-subunit (CaVβ) binds a conserved domain (the α-interaction domain (AID)) of the pore-forming CaVα1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaVβ2a. The Rem interaction module is located in a ∼130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaVβ mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaVβ2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaVβ2a interaction is lower than that of AID for CaVβ2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaVβ2a with AID. Instead, CaVβ can simultaneously associate with both Rem and CaVα1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaVβ subunits with CaVα1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem·CaVβ·AID regulatory complex without the need to disrupt CaVα1·CaVβ association or alter CaVα1 expression at the plasma membrane.

Plasma membrane targeting is essential for rem-mediated Ca2+channel inhibition

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca2+ channels and a known interacting partner for Ca2+ channel accessory β subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that CaVβ 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 β2a 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 β2a in vitro and β1b in vivo, suggesting that the C terminus does not directly participate in CaVβ association. Despite demonstrated β1b binding, Rem-(1–265) was not capable of regulating a CaV1.2-β1b channel complex, indicating that β 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 β binding and membrane localization are independent events required for channel inhibition.

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation.

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca2+ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca2+ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca2+ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of CaV1.2 (PCT), including the CB/IQ domain known to contribute to Ca2+/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the CaV1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca2+/CaM, and this effect is not due to Ca2+/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca2+ channel inhibition and slows the kinetics of Ca2+-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca2+ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca2+ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca2+ currents via modulation of Ca2+/CaM-mediated channel inactivation kinetics.

Analyses of Rem/RGK Signaling and Biological Activity

Rem (Rad and Gem related) is a member of the RGK family of Ras-related GTPases that also includes Rad, Rem2, and Gem/Kir. All RGK proteins share structural features that are distinct from other Ras-related proteins, including several nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains regulatory sites that seem to control both subcellular location and function. Rem is known to modulate two distinct signal transduction pathways, regulating both cytoskeletal reorganization and voltage-gated Ca2+ channel activity. In this chapter, we summarize the experimental approaches used to characterize the interaction of Rem with 14-3-3 proteins and Ca2+ channel beta-subunits and describe electrophysiological analyses for characterizing Rem-mediated regulation of L-type Ca2+ channel activity.

Calmodulin binding is dispensable for Rem-mediated Ca2+ channel inhibition

GTPases of the Ras-related RGK family are negative regulators of high voltage-activated (HVA) Ca2+ channel activity. In this study, we examined the role of calmodulin (CaM) association in Rem-mediated Ca2+ channel inhibition. We found that the Rem/CaM interaction is Ca2+-dependent, and that truncation of the Rem C-terminus before position 277 prevents CaM binding. Serial mutagenesis of the Rem C-terminus between residues 265 and 276 to alanine generated two mutants (RemL271A and RemL274A) that displayed reduced CaM binding, and a subset of these mutants displayed significantly lower cell periphery localization than RemWT. However, reductions in CaM association or membrane trafficking did not affect function, as all Rem mutants could completely inhibit Ca2+ channels. The Rem1–275 truncation mutant partially inhibited Ca2+ channel activity despite its inability to bind CaM. Taken together, these studies indicate that CaM association is not essential for either Rem-mediated Ca2+ channel inhibition or plasma membrane localization.

Overexpression of the Na+/K+ ATPase α2 But Not α1 Isoform Attenuates Pathological Cardiac Hypertrophy and RemodelingNovelty and Significance

© 2013 American Heart Association, Inc. Rationale: The Na+/K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+/Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance. Objective: To determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 to protect the heart. Methods and Results: Cardiac-specific transgenic mice overexpressing either NKA-α1 or NKA-α2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-α1 had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-α2 overexpression but not NKA-α1 led to significantly faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-α2 showed greater affinity for Na+ compared with NKA-α1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-α2 but not NKA-α1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca2+ removal. Conclusions: Our results suggest that the protective effect produced by increased expression of NKA-α2 on the heart after pressure overload is due to more efficient Ca2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-α1.Rationale: The Na+/K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+/Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance. Objective: To determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 to protect the heart. Methods and Results: Cardiac-specific transgenic mice overexpressing either NKA-&agr;1 or NKA-&agr;2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-&agr;1 had no protective effect, overexpression of NKA-&agr;2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-&agr;2 overexpression but not NKA-&agr;1 led to significantly faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-&agr;2 showed greater affinity for Na+ compared with NKA-&agr;1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-&agr;2 but not NKA-&agr;1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca2+ removal. Conclusions: Our results suggest that the protective effect produced by increased expression of NKA-&agr;2 on the heart after pressure overload is due to more efficient Ca2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-&agr;1.