Key points

Prevailing dogma holds that activation of the β-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type Ca_V 1.2 channel activity, resulting in increased Ca²⁺ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca_V 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca²⁺ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of Ca_V 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca²⁺ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized Ca_V 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling.

Abstract

Voltage-dependent L-type Ca_V 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the β-adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca_V 1.2 activity, resulting in increased Ca²⁺ influx into ventricular myocytes and a positive inotropic response. Ca_V 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca²⁺ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca²⁺ influx and augments myocyte Ca²⁺ transient and contraction amplitudes. We investigated whether βAR signalling could alter Ca_V 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca²⁺ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca_V 1.2-Ca_V 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of Ca_V 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal Ca_V 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of Ca_V 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR-mediated regulation of Ca_V 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal Ca_V 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.

L-type Ca_V1.2 channels are key regulators of gene expression, cell excitability and muscle contraction. Ca_V1.2 channels organize in clusters throughout the plasma membrane. This channel organization has been suggested to contribute to the concerted activation of adjacent Ca_V1.2 channels (e.g. cooperative gating). Here, we tested the hypothesis that dynamic intracellular and perimembrane trafficking of Ca_V1.2 channels is critical for formation and dissolution of functional channel clusters mediating cooperative gating. We found that Ca_V1.2 moves in vesicular structures of circular and tubular shape with diverse intracellular and submembrane trafficking patterns. Both microtubules and actin filaments are required for dynamic movement of Ca_V1.2 vesicles. These vesicles undergo constitutive homotypic fusion and fission events that sustain Ca_V1.2 clustering, channel activity and cooperative gating. Our study suggests that Ca_V1.2 clusters and activity can be modulated by diverse and unique intracellular and perimembrane vesicular dynamics to fine-tune Ca²⁺ signals.

Large-conductance Ca²⁺-activated potassium (BK_Ca) channels are key determinants of vascular smooth muscle excitability. Impaired BK_Ca channel function through remodeling of BK_Ca β1 expression and function contributes to vascular complications in animal models of diabetes. Yet, whether similar alterations occur in native vascular smooth muscle from humans with type 2 diabetes is unclear. In this study, we evaluated BK_Ca function in vascular smooth muscle from small resistance adipose arteries of non-diabetic and clinically diagnosed type 2 diabetic patients. We found that BK_Ca channel activity opposes pressure-induced constriction in human small resistance adipose arteries, and this is compromised in arteries from diabetic patients. Consistent with impairment of BK_Ca channel function, the amplitude and frequency of spontaneous BK_Ca currents, but not Ca²⁺ sparks were lower in cells from diabetic patients. BK_Ca channels in diabetic cells exhibited reduced Ca²⁺ sensitivity, single-channel open probability and tamoxifen sensitivity. These effects were associated with decreased functional coupling between BK_Ca α and β1 subunits, but no change in total protein abundance. Overall, results suggest impairment in BK_Ca channel function in vascular smooth muscle from diabetic patients through unique mechanisms, which may contribute to vascular complications in humans with type 2 diabetes.

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca²⁺ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

Elevated glucose increases vascular reactivity by promoting L-type Ca_V1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a G_s-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y₁₁, the only G_s-coupled P2Y receptor, was detected in nanometer proximity to Ca_V1.2 and PKA. FRET-based experiments revealed that the selective P2Y₁₁ agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y₁₁ inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y₁₁-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y₁₁ in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.