Unraveling the Connection: Cholesterol, Calcium Signaling, and Neurodegeneration.

Cholesterol and calcium play crucial roles as integral structural components and functional signaling entities within the central nervous system. Disruption in cholesterol homeostasis has been linked to Alzheimer's, Parkinson's, and Huntington's Disease while alterations in calcium signaling is hypothesized to be a key substrate for neurodegeneration across many disorders. Despite the importance of regulated cholesterol and calcium homeostasis for brain health there has been an absence of research investigating the interdependence of these signaling molecules and how they can tune each other's abundance at membranes to influence membrane identity. Here, we discuss the role of cholesterol in shaping calcium dynamics in a neurodegenerative disorder that arises due to mutations in the lysosomal cholesterol transporter, Niemann Pick Type C1 (NPC1). We discuss the molecular mechanisms through which altered lysosomal cholesterol transport influences calcium signaling pathways through remodeling of ion channel distribution at organelle-organelle membrane contacts leading to neurodegeneration. This scientific inquiry not only sheds light on NPC disease but also holds implications for comprehending other cholesterol-associated neurodegenerative disorders.

BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

α-Synuclein-dependent increases in PIP5K1γ drive inositol signaling to promote neurotoxicity.

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P2, which precipitates α-Syn aggregation and drives toxic increases in mitochondrial Ca2+ and reactive oxygen species leading to neuronal death. Upstream of this toxic signaling pathway is PIP5K1γ, whose abundance and localization is enhanced at the PM by α-Syn-dependent increases in ARF6. Selective inhibition of PIP5K1γ or knockout of ARF6 in neurons rescues α-Syn aggregation and cellular phenotypes of toxicity. Collectively, our data suggest that modulation of phosphoinositide metabolism may be a therapeutic target to slow neurodegeneration for PD and other related neurodegenerative disorders.

NPC1-dependent alterations in KV2.1-CaV1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease.

Lysosomes communicate through cholesterol transfer at endoplasmic reticulum (ER) contact sites. At these sites, the Niemann Pick C1 cholesterol transporter (NPC1) facilitates the removal of cholesterol from lysosomes, which is then transferred to the ER for distribution to other cell membranes. Mutations in NPC1 result in cholesterol buildup within lysosomes, leading to Niemann-Pick Type C (NPC) disease, a progressive and fatal neurodegenerative disorder. The molecular mechanisms connecting NPC1 loss to NPC-associated neuropathology remain unknown. Here we show both in vitro and in an animal model of NPC disease that the loss of NPC1 function alters the distribution and activity of voltage-gated calcium channels (CaV). Underlying alterations in calcium channel localization and function are KV2.1 channels whose interactions drive calcium channel clustering to enhance calcium entry and fuel neurotoxic elevations in mitochondrial calcium. Targeted disruption of KV2-CaV interactions rescues aberrant CaV1.2 clustering, elevated mitochondrial calcium, and neurotoxicity in vitro. Our findings provide evidence that NPC is a nanostructural ion channel clustering disease, characterized by altered distribution and activity of ion channels at membrane contacts, which contribute to neurodegeneration.

Role of Lysosomal Cholesterol in Regulating PI(4,5)P2-Dependent Ion Channel Function.

Lysosomes are central regulators of cellular growth and signaling. Once considered the acidic garbage can of the cell, their ever-expanding repertoire of functions include the regulation of cell growth, gene regulation, metabolic signaling, cell migration, and cell death. In this chapter, we detail how another of the lysosome's crucial roles, cholesterol transport, plays a vital role in the control of ion channel function and neuronal excitability through its ability to influence the abundance of the plasma membrane signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This chapter will introduce the biosynthetic pathways of cholesterol and PI(4,5)P2, discuss the molecular mechanisms through which each lipid distinctly regulates ion channels, and consider the interdependence of these lipids in the control of ion channel function.

Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

The E3 Ubiquitin Ligase CRL5 Regulates Dentate Gyrus Morphogenesis, Adult Neurogenesis, and Animal Behavior.

The dentate gyrus (DG) is an essential part of the hippocampal formation and participates in the majority of hippocampal functions. The DG is also one of the few structures in the mammalian central nervous system that produces adult-born neurons and, in humans, alterations in adult neurogenesis are associated with stress and depression. Given the importance of DG in hippocampal function, it is imperative to understand the molecular mechanisms driving DG development and homeostasis. The E3 ubiquitin ligase Cullin-5/RBX2 (CRL5) is a multiprotein complex involved in neuron migration and localization in the nervous system, but its role during development and in the adult DG remain elusive. Here, we show that CRL5 participates in mossy fiber pruning, DG layering, adult neurogenesis, and overall physical activity in mice. During DG development, RBX2 depletion causes an overextension of the DG mossy fiber infrapyramidal bundle (IPB). We further demonstrate that the increased activity in Reelin/DAB1 or ARF6 signaling, observed in RBX2 knockout mice, is not responsible for the lack of IPB pruning. Knocking out RBX2 also affects granule cell and neural progenitor localization and these defects were rescued by downregulating the Reelin/DAB1 signaling. Finally, we show that absence of RBX2 increases the number neural progenitors and adult neurogenesis. Importantly, RBX2 knockout mice exhibit higher levels of physical activity, uncovering a potential mechanism responsible for the increased adult neurogenesis in the RBX2 mutant DG. Overall, we present evidence of CRL5 regulating mossy fiber pruning and layering during development and opposing adult neurogenesis in the adult DG.

Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells.

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

Phosphoinositide transport and metabolism at membrane contact sites.

Phosphoinositides are a family of signaling lipids that play a profound role in regulating protein function at the membrane-cytosol interface of all cellular membranes. Underscoring their importance, mutations or alterations in phosphoinositide metabolizing enzymes lead to host of developmental, neurodegenerative, and metabolic disorders that are devastating for human health. In addition to lipid enzymes, phosphoinositide metabolism is regulated and controlled at membrane contact sites (MCS). Regions of close opposition typically between the ER and other cellular membranes, MCS are non-vesicular lipid transport portals that engage in extensive communication to influence organelle homeostasis. This review focuses on lipid transport, specifically phosphoinositide lipid transport and metabolism at MCS.

IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease.

Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease-causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor-stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease.

NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes.

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIß-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.

ß-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins.

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. ß-Adrenergic receptor (ßAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. ßAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, ßAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented ßAR-activated Ca2+ current augmentation. Moreover, ßAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that ßAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for ßAR-regulation of cardiac CaV1.2.

AKAP5 complex facilitates purinergic modulation of vascular L-type Ca2+ channel CaV1.2.

The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia.

Sensing of nutrients by CPT1C controls SAC1 activity to regulate AMPA receptor trafficking.

Carnitine palmitoyltransferase 1C (CPT1C) is a sensor of malonyl-CoA and is located in the ER of neurons. AMPA receptors (AMPARs) mediate fast excitatory neurotransmission in the brain and play a key role in synaptic plasticity. In the present study, we demonstrate across different metabolic stress conditions that modulate malonyl-CoA levels in cortical neurons that CPT1C regulates the trafficking of the major AMPAR subunit, GluA1, through the phosphatidyl-inositol-4-phosphate (PI(4)P) phosphatase SAC1. In normal conditions, CPT1C down-regulates SAC1 catalytic activity, allowing efficient GluA1 trafficking to the plasma membrane. However, under low malonyl-CoA levels, such as during glucose depletion, CPT1C-dependent inhibition of SAC1 is released, facilitating SAC1's translocation to ER-TGN contact sites to decrease TGN PI(4)P pools and trigger GluA1 retention at the TGN. Results reveal that GluA1 trafficking is regulated by CPT1C sensing of malonyl-CoA and provide the first report of a SAC1 inhibitor. Moreover, they shed light on how nutrients can affect synaptic function and cognition.

An anthrone-based Kv7.2/7.3 channel blocker with improved properties for the investigation of psychiatric and neurodegenerative disorders.

A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC50 = 0.16 µM in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.

Disease-associated mutations in Niemann-Pick type C1 alter ER calcium signaling and neuronal plasticity.

Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease. Given the Ca2+ hypothesis of neurodegeneration, which posits that altered Ca2+ dynamics contribute to neuropathology, we tested if disease mutations in NPC1 alter Ca2+ signaling and neuronal plasticity. We determine that NPC1 inhibition or disease mutations potentiate store-operated Ca2+ entry (SOCE) due to a presenilin 1 (PSEN1)-dependent reduction in ER Ca2+ levels alongside elevated expression of the molecular SOCE components ORAI1 and STIM1. Associated with this dysfunctional Ca2+ signaling is destabilization of neuronal dendritic spines. Knockdown of PSEN1 or inhibition of the SREBP pathway restores Ca2+ homeostasis, corrects differential protein expression, reduces cholesterol accumulation, and rescues spine density. These findings highlight lysosomes as a crucial signaling platform responsible for tuning ER Ca2+ signaling, SOCE, and synaptic architecture in health and disease.

Neuronal ER-plasma membrane junctions organized by Kv2-VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis.

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface-conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

Niemann-Pick Type C Disease Reveals a Link between Lysosomal Cholesterol and PtdIns(4,5)P2 That Regulates Neuronal Excitability.

There is increasing evidence that the lysosome is involved in the pathogenesis of a variety of neurodegenerative disorders. Thus, mechanisms that link lysosome dysfunction to the disruption of neuronal homeostasis offer opportunities to understand the molecular underpinnings of neurodegeneration and potentially identify specific therapeutic targets. Here, using a monogenic neurodegenerative disorder, NPC1 disease, we demonstrate that reduced cholesterol efflux from lysosomes aberrantly modifies neuronal firing patterns. The molecular mechanism linking alterations in lysosomal cholesterol egress to intrinsic tuning of neuronal excitability is a transcriptionally mediated upregulation of the ABCA1 transporter, whose PtdIns(4,5)P2-floppase activity decreases plasma membrane PtdIns(4,5)P2. The consequence of reduced PtdIns(4,5)P2 is a parallel decrease in a key regulator of neuronal excitability, the voltage-gated KCNQ2/3 potassium channel, which leads to hyperexcitability in NPC1 disease neurons. Thus, cholesterol efflux from lysosomes regulates PtdIns(4,5)P2 to shape the electrical and functional identity of the plasma membrane of neurons in health and disease.

Recent advances in understanding phosphoinositide signaling in the nervous system.

Polyphosphoinositides (PPIn) are essential signaling phospholipids that make remarkable contributions to the identity of all cellular membranes and signaling cascades in mammalian cells. They exert regulatory control over membrane homeostasis via selective interactions with cellular proteins at the membrane-cytoplasm interface. This review article briefly summarizes our current understanding of the key roles that PPIn play in orchestrating and regulating crucial electrical and chemical signaling events in mammalian neurons and the significant neuro-pathophysiological conditions that arise following alterations in their metabolism.

ß-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

KEY POINTS: Prevailing dogma holds that activation of the ß-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 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. CaV 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 CaV 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 CaV 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 CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the ß-adrenergic receptor (ßAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 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 ßAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the ßAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 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 CaV 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 CaV 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 CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of ßAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 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.