Excitation-transcription Coupling Research Articles

Voltage-gated calcium channels in genetic epilepsies.

Voltage-gated calcium channels (VGCC) are abundant in the central nervous system and serve a broad spectrum of functions, either directly in cellular excitability or indirectly to regulate Ca2+ homeostasis. Ca2+ ions act as one of the main connections in excitation-transcription coupling, muscle contraction and excitation-exocytosis coupling, including synaptic transmission. In recent years, many genes encoding VGCCs main α or additional auxiliary subunits have been associated with epilepsy. This review sums up the current state of knowledge on disease mechanisms and provides guidance on disease-specific therapies where applicable.

Excitation-transcription coupling, neuronal gene expression and synaptic plasticity.

Excitation-transcription coupling (E-TC) links synaptic and cellular activity to nuclear gene transcription. It is generally accepted that E-TC makes a crucial contribution to learning and memory through its role in underpinning long-lasting synaptic enhancement in late-phase long-term potentiation and has more recently been linked to late-phase long-term depression: both processes require de novo gene transcription, mRNA translation and protein synthesis. E-TC begins with the activation of glutamate-gated N-methyl-D-aspartate-type receptors and voltage-gated L-type Ca2+ channels at the membrane and culminates in the activation of transcription factors in the nucleus. These receptors and ion channels mediate E-TC through mechanisms that include long-range signalling from the synapse to the nucleus and local interactions within dendritic spines, among other possibilities. Growing experimental evidence links these E-TC mechanisms to late-phase long-term potentiation and learning and memory. These advances in our understanding of the molecular mechanisms of E-TC mean that future efforts can focus on understanding its mesoscale functions and how it regulates neuronal network activity and behaviour in physiological and pathological conditions.

Neuronal ER-plasma membrane junctions couple excitation to Ca2+-activated PKA signaling

Junctions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are specialized membrane contacts ubiquitous in eukaryotic cells. Concentration of intracellular signaling machinery near ER-PM junctions allows these domains to serve critical roles in lipid and Ca2+ signaling and homeostasis. Subcellular compartmentalization of protein kinase A (PKA) signaling also regulates essential cellular functions, however, no specific association between PKA and ER-PM junctional domains is known. Here, we show that in brain neurons type I PKA is directed to Kv2.1 channel-dependent ER-PM junctional domains via SPHKAP, a type I PKA-specific anchoring protein. SPHKAP association with type I PKA regulatory subunit RI and ER-resident VAP proteins results in the concentration of type I PKA between stacked ER cisternae associated with ER-PM junctions. This ER-associated PKA signalosome enables reciprocal regulation between PKA and Ca2+ signaling machinery to support Ca2+ influx and excitation-transcription coupling. These data reveal that neuronal ER-PM junctions support a receptor-independent form of PKA signaling driven by membrane depolarization and intracellular Ca2+, allowing conversion of information encoded in electrical signals into biochemical changes universally recognized throughout the cell.

Abstract P3142: Engineering A Genetically Encoded Enhancer Of L-type Ca 2+ Currents

L-type calcium channels (LTCC) play a crucial role as they serve as vital portals for Ca 2+ entry into cardiac myocytes, neurons, and other physiological systems. Altered LTCC function is linked to various pathologies including cardiac arrhythmias and neurodevelopmental disorders. In particular, genetic studies have identified loss-of-function mutations in Ca V 1.2 channels in patients with shortened QT interval and Brugada Syndrome, as well as neuropsychiatric disorders. Given the broad physiological importance of LTCC in various tissues, conventional pharmacological agonists of LTCC may yield off-target effects and toxicity. Therefore, new approaches to upregulate LTCC function in a cell-type dependent manner is sought after. To do so, we here develop a genetically encoded enhancer of calcium channels (GeeCC, “geek”) by leveraging both a cardiac specific L-type channel modulator, leucine rich repeat containing protein 10 (Lrrc10), and selective nanobodies that target the LTCC complex. Specifically, Lrrc10 co-expression markedly upregulates LTCC currents. Furthermore, mutations in Lrrc10 are linked to cardiac diseases, including cardiomyopathies. Through extensive structure-function analysis, we identified a minimal action domain within Lrrc10 that is critical for functional modulation of LTCC. However, as this segment alone has a low affinity for the channel complex, its expression alone yielded minimal changes in Ca 2+ current amplitude. However, fusion of this Lrrc10 domain to a nanobody (nb.F3) that targets the Ca 2+ channel beta subunit resulted in a significant increase in the LTCC current amplitude. In depth analysis revealed exquisite selectivity of GeeCC for Ca V 1.2/Ca V 1.3 channels. In mouse cardiomyocytes, deployment of GeeCC via adenovirus enhances endogenous L-type currents when compared to uninfected controls or cardiomyocytes infected with nanobodies alone. Additionally, in neurons, co-expression of GeeCC enhanced excitation-transcription coupling. In all, we have developed a genetically-encoded approach to selectively enhance L-type currents. This strategy may enable new insights into cardiovascular and neuronal physiology and pathophysiology, and may have potential therapeutic applications.

Microglia sense and suppress epileptic neuronal hyperexcitability

Microglia are the resident immune cells of the central nervous system, undertaking surveillance role and reacting to brain homeostasis and neurological diseases. Recent studies indicate that microglia modulate epilepsy-induced neuronal activities, however, the mechanisms underlying microglia-neuron communication in epilepsy are still unclear. Here we report that epileptic neuronal hyperexcitability activates microglia and drives microglial ATP/ADP hydrolyzing ectoenzyme CD39 (encoded by Entpd1) expression via recruiting the cAMP responsive element binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) from cytoplasm to the nucleus and binding to CREB. Activated microglia in turn suppress epileptic neuronal hyperexcitability in a CD39 dependent manner. Disrupting microglial CREB/CRTC1 signaling, however, decreases CD39 expression and diminishes the inhibitory effect of microglia on epileptic neuronal hyperexcitability. Overall, our findings reveal CD39-dependent control of epileptic neuronal hyperexcitability by microglia is through an excitation-transcription coupling mechanism.

Clustering of CaV 1.3 L-type calcium channels by Shank3.

Clustering of L-type voltage-gated Ca2+ channels (LTCCs) in the plasma membrane is increasingly implicated in creating highly localized Ca2+ signaling nanodomains. For example, neuronal LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca2+ concentrations within a nanodomain close to the channel, without requiring bulk Ca2+ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the CaV 1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed CaV 1.3 α1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble complexes containing multiple CaV 1.3 α1 subunits under basal conditions. Moreover, CaV 1.3 LTCC complex formation was facilitated by CaV β subunits (β3 and β2a), which also interact with Shank3. Shank3 interactions with CaV 1.3 LTCCs and multimeric CaV 1.3 LTCC complex assembly were disrupted following the addition of Ca2+ to cell lysates, perhaps simulating conditions within an activated CaV 1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized CaV 1.3 LTCC clusters under basal conditions, but not after Ca2+ channel activation. Live cell imaging studies also revealed that Ca2+ influx through LTCCs disassociated Shank3 from CaV 1.3 LTCCs clusters and reduced the CaV 1.3 cluster intensity. Deletion of the Shank3 PDZ domain prevented both binding to CaV 1.3 and the changes in multimeric CaV 1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized CaV 1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions.

Exercise and calcium in the heart

Cardiomyocyte Ca2+ dictates cardiac contraction, via excitation-contraction coupling and excitation-transcription coupling. Adaptation to these processes also majorly contributes to enhanced contractile function and capacity following exercise training. Cytoplasmic Ca2+ release controls sarcomeric contraction, with important modulation by the voltage-sensitive plasma membrane L-type Ca2+ channel and the Ryanodine receptor, as well as the Sarcoplasmic reticulum Ca2+ ATPase. Exercise training increases and enhances these excitation-contraction coupling sub-processes, in a manner that increases and enhances cardiac contraction. Also, adaptation to exercise training further includes myofilament Ca2+ sensitization. Then there are several aspects linked to post-exercise training cardiomyocyte Ca2+ handling that remain speculative and inconclusive, but could if proven true be of special importance. This includes Ca2+-linked muscle-specific gene transcription to alter cell architecture and size, and it includes the scenario whereby Ca2+ cycling and adaptations may alter arrhythmogenicity. These aspects of cardiac Ca2+ adaptations to exercise training are discussed in this review article.

Fibroblast growth factor 21 is expressed and secreted from skeletal muscle following electrical stimulation via extracellular ATP activation of the PI3K/Akt/mTOR signaling pathway.

Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a "myokine", synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as "Excitation-Transcription coupling". eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.

Towards next-generation optogenetic actuators of voltage-gated Ca2+ channels.

CaV1 channels (CaV1.2/1.3) are fundamentally involved in the normal function and pathophysiology of the heart and the brain. These channels convey Ca2+ influx that sculpts action potentials, triggers muscle contraction, and mediates excitation-transcription coupling. To orchestrate these diverse functions, CaV1 activity is fine-tuned by multiple regulatory processes, including Ca2+-dependent inactivation (CDI), which is mediated by the Ca2+-binding protein, calmodulin (CaM). Disruption of CaM regulation is linked to cardiac arrhythmias and neurodevelopmental disorders. However, the precise role of this modulation in various physiological settings is not fully understood. In part, this gap in understanding stems from a lack of approaches to dynamically alter CDI. Here, we engineer a novel optogenetic tool to acutely prevent CDI. To do so, we undertook a biomimetic approach by leveraging naturally occurring CaV1 regulatory mechanisms that modify CDI. Specifically, SH3 and cysteine-rich-domain (stac) protein was recently found to selectively prevent CaV1 CDI. Our previous work identified a short amino acid segment of stac called the U-domain that strongly inhibits CDI. Here, we attached the stac3 U-domain to a light-sensitive Lov2 domain to optically switch its accessibility for modulating CaV1 channels, yielding an actuator known as LovU-CaV1. Whole-cell electrophysiological recordings showed that co-expression of LovU-CaV1 resulted in low basal inhibition of CDI of CaV1.2. However, upon light activation of LovU-CaV1, we observed a rapid reduction in CDI that was then reversible. LovU-CaV1 provides a new avenue to optically manipulate endogenous Ca channels in both the heart and the brain. Future studies will employ this tool to dissect the importance of L-type channel inactivation in both cardiomyocytes and in neurons.

Excitation-transcription coupling in smooth muscle is associated with vascular remodeling

Excitation-transcription coupling in smooth muscle is associated with vascular remodeling

Current updates on arrhythmia within Timothy syndrome: genetics, mechanisms and therapeutics.

Timothy syndrome (TS), characterised by multiple system malfunction especially the prolonged corrected QT interval and synchronised appearance of hand/foot syndactyly, is an extremely rare disease affecting early life with devastating arrhythmia. In this work, firstly, the various mutations in causative gene CACNA1C encoding cardiac L-type voltage-gated calcium channel (LTCC), regard with the genetic pathogeny and nomenclature of TS are reviewed. Secondly, the expression profile and function of CACNA1C gene encoding Cav1.2 proteins, and its gain-of-function mutation in TS leading to multiple organ disease phenotypes especially arrhythmia are discussed. More importantly, we focus on the altered molecular mechanism underlying arrhythmia in TS, and discuss about how LTCC malfunction in TS can cause disorganised calcium handling with excessive intracellular calcium and its triggered dysregulated excitation-transcription coupling. In addition, current therapeutics for TS cardiac phenotypes including LTCC blockers, beta-adrenergic blocking agents, sodium channel blocker, multichannel inhibitors and pacemakers are summarised. Eventually, the research strategy using patient-specific induced pluripotent stem cells is recommended as one of the promising future directions for developing therapeutic approaches. This review updates our understanding on the research progress and future avenues to study the genetics and molecular mechanism underlying the pathogenesis of devastating arrhythmia within TS, and provides novel insights for developing therapeutic measures.

Selective posttranslational inhibition of CaVβ1-associated voltage-dependent calcium channels with a functionalized nanobody.

Ca2+ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca2+ influx, to mediate disparate functions is diversity of HVACC pore-forming α1 and auxiliary CaVβ1-CaVβ4 subunits. Selective CaVα1 blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on CaVβ isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of CaVβ isoforms. Here, we identify a nanobody (nb.E8) that selectively binds CaVβ1 SH3 domain and inhibits CaVβ1-associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through CaVβ1-reconstituted CaV1/CaV2 and native CaV1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca2+ influx and excitation-transcription coupling in hippocampal neurons, but was inert against CaVβ2-associated CaV1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of CaVβ1 signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.

Autism associated mutations in β2 subunit of voltage-gated calcium channels constitutively activate gene expression

Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α11.2, or β2d, are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavβ2a corresponding to autistic related mutations Cavβ2dS197F and Cavβ2dG167S in the human Cavβ2d, activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α11.2 and α2δ with Cavβ2aS143F or Cavβ2aG113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α11.2G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavβ2a autistic mutants is consistent with Cavβ2/Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα11.2/Cavβ2 . Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca2+ channel mediated ASD and other neurodevelopmental disorders.

Nuanced Interactions between AKAP79 and STIM1 with Orai1 Ca2+ Channels at Endoplasmic Reticulum-Plasma Membrane Junctions Sustain NFAT Activation.

A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca2+/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca2+ store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca2+ channels, enabling Ca2+ nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca2+ entry. We also show that Ca2+ nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.

Real-time imaging of Arc/Arg3.1 transcription ex vivo reveals input-specific immediate early gene dynamics

The ability of neurons to process and store salient environmental features underlies information processing in the brain. Long-term information storage requires synaptic plasticity and regulation of gene expression. While distinct patterns of activity have been linked to synaptic plasticity, their impact on immediate early gene (IEG) expression remains poorly understood. The activity regulated cytoskeleton associated (Arc) gene has received wide attention as an IEG critical for long-term synaptic plasticity and memory. Yet, to date, the transcriptional dynamics of Arc in response to compartment and input-specific activity is unclear. By developing a knock-in mouse to fluorescently tag Arc alleles, we studied real-time transcription dynamics after stimulation of dentate granule cells (GCs) in acute hippocampal slices. To our surprise, we found that Arc transcription displayed distinct temporal kinetics depending on the activation of excitatory inputs that convey functionally distinct information, i.e., medial and lateral perforant paths (MPP and LPP, respectively). Moreover, the transcriptional dynamics of Arc after synaptic stimulation was similar to direct activation of GCs, although the contribution of ionotropic glutamate receptors, L-type voltage-gated calcium channel, and the endoplasmic reticulum (ER) differed. Specifically, we observed an ER-mediated synapse-to-nucleus signal that supported elevations in nuclear calcium and, thereby, rapid induction of Arc transcription following MPP stimulation. By delving into the complex excitation-transcription coupling for Arc, our findings highlight how different synaptic inputs may encode information by modulating transcription dynamics of an IEG linked to learning and memory.

Local Ca2+ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells.

An increase in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-sensitive enzymes such as Ca2+/calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca2+ channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca2+]i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca2+ signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca2+ chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca2+ events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca2+ signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.

Roles of ATP and SERCA in the Regulation of Calcium Turnover in Unloaded Skeletal Muscles: Current View and Future Directions.

A decrease in skeletal muscle contractile activity or its complete cessation (muscle unloading or disuse) leads to muscle fibers’ atrophy and to alterations in muscle performance. These changes negatively affect the quality of life of people who, for one reason or another, are forced to face a limitation of physical activity. One of the key regulatory events leading to the muscle disuse-induced changes is an impairment of calcium homeostasis, which leads to the excessive accumulation of calcium ions in the sarcoplasm. This review aimed to analyze the triggering mechanisms of calcium homeostasis impairment (including those associated with the accumulation of high-energy phosphates) under various types of muscle unloading. Here we proposed a hypothesis about the regulatory mechanisms of SERCA and IP3 receptors activity during muscle unloading, and about the contribution of these mechanisms to the excessive calcium ion myoplasmic accumulation and gene transcription regulation via excitation–transcription coupling.

A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation–transcription coupling

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation–transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

On the molecular mechanism of excitation–transcription coupling in skeletal muscle

An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation-transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.

Regulation of neuronal excitation–transcription coupling by Kv2.1-induced clustering of somatic L-type Ca2+ channels at ER-PM junctions

In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation-transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation-transcription coupling. The voltage-gated K+ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum-plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation-transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1's C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum-plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.