Totally Rad? The long and winding road to understanding Ca_V1.2 regulation

It has been clear for over 100 years that Ca²⁺ and catecholamines perform central roles necessary for sustaining and modulating heart function. However, despite years of study, the cellular and molecular mechanisms underpinning adrenergic receptor agonist activation of voltage-gated Ca²⁺ current have been far less certain. The voltage-gated Ca²⁺ channel Ca_V1.2 is the primary pathway for extracellular Ca²⁺ to enter myocardium. This process is by necessity tightly controlled, in part, because of the steep extracellular – intracellular gradient, where extracellular Ca²⁺ concentration is 10,000 × greater than resting intracellular Ca²⁺ concentration. To avoid pathological intracellular Ca²⁺ overload, the Ca_V1.2 current (I_Ca) is meted out in discrete pulses, substantially under the control of cell membrane voltage and adrenergic tone, to direct excitation-contraction coupling and inotropy, and to influence metabolism and gene transcription. Unfortunately, in tough times, loss of normal Ca²⁺ homeostasis contributes to myocardial dysfunction, arrhythmias and death. Thus, understanding the control points for operating this system is an important goal for cardiovascular research. Work published in this issue of Circulation Research by Papa et al.¹ provides new genetic insights into a mechanism for how Ca_V1.2 operates with a higher probability of opening (Po) in heart failure,² a circumstance contributing to arrhythmia and, perhaps, to heart failure itself.

Despite the undisputed centrality of I_Ca to myocardial biology, and sustained and substantial research efforts, this system has been reluctant to divulge its secrets. On one hand, the core biophysical details of the cell membrane conductance changes that orchestrate each myocardial action potential are substantially understood. On the other hand, the mechanisms whereby protein kinase A mediated phosphorylation leads to increased I_Ca have been more resistant to discovery. The twin revolutions of genetics and molecular biology identified the pore forming α subunit³ and the, so called, auxiliary subunits, including the β subunits,⁴ for Ca_V1.2. Based on this body of knowledge it seemed likely that it would be, more or less, straightforward to identify the key protein kinase A (PKA) catalyzed phosphorylation sites on the α and or β subunits, and that these sites would explain how catecholamines modify Ca_V1.2 to augment I_Ca. At first, the published literature seemed to support this optimistic prediction. Multiple phosphorylation sites were identified on the α and β subunits that appeared to directly or indirectly affect Ca_V1.2 responses to adrenergic signaling. However, none of these withstood the scrutiny of protracted research.⁵ In some cases, these sites seemed to play a role that was cell or context specific, but not applicable to cardiomyocytes, whereas other observations were not repeatable by a broader community of researchers. More recently, a series of important collaborative studies, mostly led by the Marx group, have provided exciting data that together provide a robust framework to understand how adrenergic receptor agonist stimulation modifies Ca_V1.2 to increase I_Ca in heart.

In order to place the most recent findings of Papa et ai. into context it is worthwhile to briefly review this foundation of experimental approaches and earlier findings. The Marx group developed a flexible tool kit with the power to interrogate candidate sites on Ca_V1.2 α and β subunits in adult cardiomyocytes. They engineered transgenic mice with inducible myocardial expression of a dihydropyridine-resistant Ca_V1.2 α subunit.⁶ Aside from its relative resistance to dihydropyridine class Ca²⁺ channel antagonists, this mutant channel was verified to operate in other ways similarly to its wild type counterpart. The dihydropyridine resistance of the mutant channels permitted selective pharmacological ‘silencing’ of the wild type channels, allowing the investigators to measure the consequences of mutations on I_Ca in the context of a fully differentiated adult cardiomyocyte. A series of studies using this mouse model, where various PKA candidate sites were mutated, revealed that multiple residues and segments of the α subunit were dispensable, but α-β subunit binding was essential for adrenergic receptor activation mediated increases in I_Ca.⁷ More recently, Liu et al.⁸ mutated each serine and threonine (51 in total) with the potential to contribute to a PKA consensus site in the α subunit and found these PKA resistant α subunits assembled into Ca_V1.2 with a normal adrenergic receptor agonist I_Ca responses. Based on this seemingly definitive evidence that the α subunit was not a direct target for PKA, they used proximity labeling (with ascorbate peroxidase [APEX2] and a biotin interacting epitope fused to the α or β subunits) to interrogate nearby (~20 nm) proteins with the potential to confer adrenergic receptor mediated stimulation to I_Ca.⁸ These studies identified Rad as a close neighbor of the α and β subunits under basal conditions, but showed Rad’s proximity to these Ca_V1.2 components vanished after adrenergic receptor stimulation with isoproterenol, consistent with Rad acting as a tonic inhibitor of I_Ca. Rad, a GTP binding protein, was in intriguing ‘hit’, as it and other RGK family members (Rad, Rem, Rem2 and Gem/Kir) were known to inhibit voltage-gated Ca²⁺ channels, including Ca_V1.2.⁹ The Liu et al. studies⁸ revealed key PKA phosphorylation sites on Rad that uncoupled Rad from the Ca_V1.2 α-β complex to confer adrenergic receptor agonist mediated I_Ca disinhibition (i.e. augmentation). Taken together, these studies established that the Ca_V1.2 α subunit was almost certainly not a direct target for adrenergic receptor/PKA pathway driven activation, that the β subunit was necessary and that a key phosphorylation target was Rad, which in the absence of PKA catalyzed phosphorylation, acted as a tonic I_Ca suppressor. Stated another way, catecholamines increase I_Ca, a central event in fight or flight physiology, by phosphorylating Rad and releasing I_Ca from tonic Rad inhibition.

This newest paper by Papa et al.¹ provides new evidence for understanding how Ca_V1.2 opening probability is affected through the expression of a splice variant exon 9*, which is over-represented in failing human and rodent hearts. It has been known for some time that Ca_V1.2 channels exhibit enhanced opening probability in cardiomyocytes isolated from failing human hearts.² The increased opening probability of these Ca_V1.2 channels is consequential because it contributes to cell membrane hyperexcitability, afterdepolarizations and arrhythmias. PKA and calmodulin kinase II (CaMKII)¹⁰ are both capable of driving this hyperactive Ca_V1.2 behavior, and CaMKII expression and activity are increased in failing hearts. But these kinase signals increase Ca_V1.2 opening probability from healthy and failing myocardium. Intriguingly, expressed exon 9* is a component of the I-II linker domain of Ca_V1.2, part of the elaborate intracellular protein architecture involved in β subunit binding. In order to test for a functional consequence of this relatively over-expressed exon, Papa et al. transgenically over-expressed the dihydropyridine resistant Ca_V1.2 engineered with exon 9* in mouse hearts. After isolating ventricular myocytes and silencing the endogenous Ca_V1.2 channels, they found that I_Ca from CaV1.2 channels expressing the 9* exon was activated at a more negative cell membrane potentials and exhibited higher Po compared to wild type channels. These hyperactivated Ca_V1.2 behaviors are normally present in catecholamine and CaMKII stimulated channels, and appeared similar to the phenotypes observed in failing human hearts.

In my view, these findings are both interesting and important, but also raise critical unanswered questions. Despite the power of the transgenic mouse model with conditional over-expression of dihydropyridine resistant ion channels, we don’t know anything about the in vivo consequences of exon 9* over-expression, or the regulatory steps that favor expression of this splice variant in sick hearts. An important short coming of this transgenic model is that induction of the transgenic channel in the absence of silencing of the native channels creates an established pathological scenario of excessive Ca_V1.2. Furthermore, it is not possible to use dihydropyridines in vivo at a concentration required to silence the endogenous wild type channels because of off target consequences to blood pressure. Because of these limitations, Papa et al. do not tell us if increased exon 9* expression is a cause of cardiomyopathy, or predisposes to arrhythmias; we don’t know if interventions that enhance the α subunit I-II linker flexibility, as represented by the GGG mutant, could protect against heart failure and arrhythmias. Thus, we do not yet know about the in vivo consequences of enhanced Ca_V1.2 exon 9* expression. Enhanced activity of another Ca²⁺ channel, the intracellular RyR2, is linked to heart failure and arrhythmias,¹¹ but patients with CPVT (catecholaminergic ventricular tachycardia), mostly due to genetic defects that lead to hyperactive, ‘leaky’ RyR2 do not exhibit important cardiomyopathy. Thus, by analogy, it could be that hyperactive Ca_V1.2 contributes to arrhythmia, but is insufficient to cause cardiomyopathy. However, Ca_V1 is part of a key Ca²⁺ pathway for controlling transcription in neurons¹² and it is possible that Ca_V1.2 plays a similar role in cardiomyocytes and that this pathway could be adaptive or maladaptive. For example, it could be that exon 9* contributes to a short-term compensation for failing myocardium, but, similar to pharmacological inotropic therapies, over-representation of exon 9* will ultimately promote arrhythmias and myocardial injury. We will need to wait for new models suitable for in vivo use to measure and comprehend the physiological and disease consequences of exon 9*.

Finally, Papa et al. engineered the Ca_V1.2 intracellular I-II linker with a hyperflexible GGG sequence and found that, in contrast to the modeled rigidity of the exon 9* linker, the GGG channels were nearly unresponsive to adrenergic receptor pathway activation despite binding the β subunit. This finding is intriguing and inspires speculation that the intracellular I-II linker somehow acts as a lever or a pull switch to moderate and enhance the opening probability of Ca_V1.2. If true, such a concept might be expanded to other ion channels and have myriad implications for the potential of various intracellular binding partners to affect ion channel gating – by pulling at heart strings.