Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress

Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca²⁺ handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na⁺/Ca²⁺ exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.In working ventricular myocytes, normal excitation-contraction (E-C) coupling requires precise communication between voltage-gated L-type Ca²⁺ channels (Cav1.2) located in clusters within transverse (T)-tubules and, less frequently, on the plasmalemma, and Ca²⁺ release channels/ryanodine receptor channels (RyRs) that are also clustered on the junctional sarcoplasmic reticulum (jSR) membrane (1–4). In normal hearts, flat jSR cisternae containing a continuous row of polymerized calsequestrin (CsQ2) either wrap around a T-tubule segment or abut against the plasmalemma (5, 6) and are coupled to the surface membranes via apposed clusters of RyR2 and Cav1.2 (7). These junctional sites are called dyads. However, although the jSR cisternae constitute a single continuous compartment, the clusters of RyR2 do not occupy the whole jSR surface but are in smaller groups (8, 9). Hence, each dyad is composed of several smaller RyR2/Cav1.2 complexes, also called couplons. Functional interaction between Cav1.2 and RyR2 at these sites ensure synchronous SR Ca²⁺ release and coordinated contraction (1, 10, 11). There is evidence that impaired cardiac E-C coupling/Ca²⁺ handling is a key mediator of heart failure (12, 13). One underlying mechanism for the defective Ca²⁺ release is the progressive loss of T-tubule network organization and of the relationship between RyR2 and Cav1.2 (14–16). Therefore, preventing loss of jSR/T-tubule junctions and of T-tubule organization may represent a new strategy for therapeutic intervention in heart failure.In normal cardiomyocytes, the formation of dyads requires junctophilin 2 (JP2), a structural protein that provides a physical connection between the T-tubule and SR membranes (17). JP2’s eight N-terminal “membrane occupation and recognition nexus” domains bind to the plasmalemma (T-tubules), and its C-terminal transmembrane domain tethers the opposite end to the SR membrane (17). Decreased JP2 levels have been observed in human heart failure patients and in failing hearts from animal models of cardiac disease (16, 18–22). Knockdown of JP2 results in acute heart failure that is associated with the loss of junctional membrane complex, disrupted T-tubule organization, and Ca²⁺ handling dysfunction (23). In addition, embryonic myocytes with JP2 deficiency have defective cardiac dyads, including more SR segments with no T-tubule couplings as well as reduced intracellular Ca²⁺ transients (17). These data collectively suggest that loss of JP2 contributes to the functional defects in heart failure. Therefore, interesting questions are: Is the JP2 deficiency effect linked to the resultant disruption of jSR/T-tubule junctions and of T-tubule network integrity, as suggested by previous findings (16–18, 23)? Conversely, could exogenous overexpression of JP2 in cardiomyocytes improve Ca²⁺ handling and protect against the development of heart failure?To answer this question, we generated transgenic mice with cardiac-specific overexpression of JP2. Moderate overexpression of JP2 led to a significant increase in the junctional coupling area between T-tubule and SR membrane, but surprisingly, it did not enhance cardiac function or increase SR Ca²⁺ release at baseline. However, interestingly, JP2-overexpressing mice were resistant to left ventricular pressure overload-induced heart failure, demonstrating that JP2 overexpression is protective. These data suggest that preventing the loss of JP2 could be a potential therapeutic strategy for heart failure treatment.     

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca²⁺ signals is due to a signal-enhancing analysis of the Ca²⁺ activity. The rapid stimulation-evoked Ca²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.Brain function emerges from signaling in and between neurons and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1–5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca²⁺ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7–10). Ca²⁺ responses are of interest because intracellular Ca²⁺ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca²⁺-dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca²⁺ changes (11). Astrocytic Ca²⁺ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8, 10, 12, 13). Furthermore, uncaging of Ca²⁺ in astrocytes triggers vascular responses in brain slices through specific Ca²⁺-dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca²⁺ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16, 17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca²⁺ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca²⁺ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca²⁺ responses were brief enough to be a direct consequence of synaptic excitation and correlated with stimulation-induced hemodynamic responses. Fast Ca²⁺ responses in astrocyte end-feet preceded the onset of dilatation in adjacent vessels by hundreds of milliseconds. This finding might suggest that communication at the gliovascular interface contributes considerably to neurovascular coupling.     

From the Cover: Mitochondrial calcium overload is a key determinant in heart failure

Calcium (Ca²⁺) released from the sarcoplasmic reticulum (SR) is crucial for excitation–contraction (E–C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca²⁺ is essential for optimal function of these organelles. However, Ca²⁺ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca²⁺ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca²⁺ leak causes mitochondrial Ca²⁺ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca²⁺ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca²⁺ leak, we found that leaky RyR2 channels result in mitochondrial Ca²⁺ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca²⁺ overload and dysfunction in HF.Type 2 ryanodine receptor/Ca²⁺ release channel (RyR2) and type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) are the major intracellular Ca²⁺ release channels in the heart (1–3). RyR2 is essential for cardiac excitation–contraction (E–C) coupling (2), whereas the role of IP3R2 in cardiomyocytes is less well understood (3). E–C coupling requires energy in the form of ATP produced primarily by oxidative phosphorylation in mitochondria (4–8).Both increased and reduced mitochondrial Ca²⁺ levels have been implicated in mitochondrial dysfunction and increased reactive oxygen species (ROS) production in heart failure (HF) (6, 7, 9–17). Albeit Ca²⁺ is required for activation of key enzymes (i.e., pyruvate dehydrogenase phosphatase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the tricarboxylic acid (also known as Krebs) cycle (18, 19), excessive mitochondrial Ca²⁺ uptake has been associated with cellular dysfunction (14, 20). Furthermore, the exact source of mitochondrial Ca²⁺ has not been clearly established. Given the intimate anatomical and functional association between the sarcoplasmic reticulum (SR) and mitochondria (6, 21, 22), we hypothesized that SR Ca²⁺ release via RyR2 and/or IP3R2 channels in cardiomyocytes could lead to mitochondrial Ca²⁺ accumulation and dysfunction contributing to oxidative overload and energy depletion.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca²⁺ transients, decreased intracellular Ca²⁺ leak and increased sarcoplasmic reticulum (SR) Ca²⁺ load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca²⁺ release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (P_o). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca²⁺ leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.Age-dependent muscle weakness is a leading cause of morbidity due to frailty, loss of independence, and physical disability that is associated with increased risk of falls and fractures (1, 2). In geriatric populations age-dependent muscle weakness, characterized both by loss of lean muscle mass (sarcopenia) and reduced skeletal muscle function (3–5), has been estimated to affect 30–50% of 80-y-olds (1, 2, 4).The ‘free radical theory’ of aging, first proposed in 1956 by Harman (6), states that an underlying mechanism of age-dependent pathology is the accumulation of partially reduced forms of oxygen (7, 8), collectively known as reactive oxygen species (ROS). Mitochondria are a major source of cellular ROS (7, 9) and have been proposed to play a key role in age-dependent loss of skeletal muscle function (3, 7, 10), likely through the production of oxidative damage (11, 12). However, the molecular mechanisms underlying this process have not been fully determined.Skeletal muscle contraction is dependent upon release of intracellular Ca²⁺ via the sarcoplasmic reticulum (SR) Ca²⁺ release channel, ryanodine receptor 1 (RyR1). Following membrane depolarization, voltage-sensing Ca²⁺ channels in the transverse tubules (Cav1.1) activate RyR1 and the ensuing rise in cytoplasmic [Ca²⁺] causes muscle contraction via the actin-myosin cross bridge cycle (13). The RyR1 is a homotetrameric protein complex composed of four monomers, kinases, a phosphatase (PP1), phosphodiesterase (PDE4D3), calmodulin, and the RyR1 channel-stabilizing subunit calstabin1 (FK506 binding protein 12, FKBP12) (14). Posttranslational modifications of the channel, including oxidation, cysteine-nitrosylation, and cAMP-dependent protein kinase A-mediated phosphorylation have been linked to impaired Ca²⁺ handling and perturbed contractility in chronic muscle fatigue, heart failure and muscular dystrophy (13–15). Furthermore, we have recently reported that both oxidation of RyR1 and the subsequent intracellular Ca²⁺ leak underlie the age-dependent reduction in skeletal muscle specific force (10). Acute induction of RyR1-mediated SR Ca²⁺ leak with rapamycin, which competes the channel-stabilizing subunit, calstabin1, off from RyR1 (14, 16), resulted in defective mitochondrial function associated with elevated free radical production (10). However, the role of mitochondrial ROS in age-dependent reduction in skeletal muscle function and exercise capacity has not been elucidated.Recently, there have been numerous efforts to study mitochondria-derived free radicals in health and lifespan by experimentally expressing catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen, in the mitochondria. This has been done using in vitro models (17), adeno-associate viral vectors (AAV) (18), and most recently by genetically engineering its overexpression in mice (19). These transgenic mice, MCat mice, in which the human catalase is targeted to and overexpressed in mitochondria, display a 10–20% increase in maximum and median lifespan (19), reduced age-related insulin resistance (20), and attenuated energy imbalance.Because mitochondrial targeted overexpression of catalase results in reduced mitochondrial ROS (19, 20), we used the MCat mouse model to investigate the relationship between antioxidant activity and skeletal muscle aging and subsequent functional decline. Aged MCat mice displayed improved voluntary exercise, increased skeletal muscle specific force, increased tetanic Ca²⁺ transients, reduced intracellular Ca²⁺ leak and increased SR Ca²⁺ load compared with age-matched wild-type (WT) littermates. RyR1 channels from aged MCat mice were less oxidized, depleted of calstabin1 and exhibited increased single channel open probability (P_o). Furthermore, pharmacological application of an antioxidant to aged WT RyR1 reduced SR Ca²⁺ leak. We have therefore identified mitochondria as a source of ROS involved in the RyR1 oxidation underlying age-associated skeletal muscle dysfunction.     

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca²⁺ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.Two-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca²⁺ and Na⁺ channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca²⁺/Na⁺ channel with cytosolic NH₂ and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membrane-spanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2–7) that may support such a lengthy evolutionary pedigree.In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca²⁺ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4–6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca²⁺ release from “acidic stores.” The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na⁺ channels regulated by the endolysosomal phosphoinositide PI(3,5)P₂. Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactome establishes a useful community resource as a “rosetta stone” for interrogating the cell biology of TPCs and their regulation. The dataset reveals a predomination of links between TPCs and effectors controlling membrane organization and trafficking, relevant for disease states involving lysosomal proliferation where TPC functionality may be altered (14).     

Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm

Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca_v3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca²⁺ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca²⁺ channels in the medial septal GABAergic projection neurons in this behavior.When confronted with an unfamiliar environment, or physical or social objects, animals often exhibit behavior patterns that can broadly be termed exploration, such as moving around the environment, touching or sniffing novel objects, and interacting with social stimuli (1). Social exploration involves complex processes that differ from those involved in the nonsocial exploration (2). Several distinctions were proposed to categorize the different forms of nonsocial exploratory behaviors from a motivational perspective (3). Behaviorally, two types of nonsocial exploration are observed in rodents and humans (3–5): object exploration and spatial or environmental exploration in the absence of objects. Object exploration is the behavior to explore discrete novel objects. This activity is elicited and sustained by the physical presence of an object. Several types of preference or “novelty” tests have been developed to investigate object exploration in rodents (3, 5–7). Environmental or spatial exploration in the absence of objects refers to the inquisitive activity of an animal in a new space, where the eliciting and sustaining stimulus is the “place” itself. Various forms of open-field tests have been used to investigate environmental or spatial exploration in rodents (3, 5, 8). Experimentally, however, the distinction can be less obvious because both can occur together (4, 7–9). Spatial exploration is suggested to be hippocampal-dependent (10)—although that is controversial (11)—whereas object exploration is suggested to be hippocampal-independent (12). Thus, it is still a matter of debate whether animal exploration belongs to a unitary category or not (9). To resolve this issue, neural definitions of these two previously proposed exploratory behaviors are needed.Interestingly, the medial septum (MS), where Ca_v3.1 T-type Ca²⁺ channels are highly expressed (13), is suggested to be critical for exploratory behaviors (5, 14–16). Moreover, the MS is also the nodal point for ascending afferent systems involved in the generation of hippocampal theta rhythms, the largest synchronous oscillatory signals in the mammalian brain, which are implicated in diverse brain functions (17, 18). Although the heterogeneity of hippocampal theta rhythms has long been under debate (19), recent studies based on genetic mutations in mice and optogenetics provide strong support for theta rhythm heterogeneity (20–22). However, their exact behavioral correlates are still debated. Ca_v3.1 Ca²⁺ channels play an important role in diverse behaviors, as well as the generation of physiologic and pathophysiologic brain rhythms (23). Notably, T-type, low-threshold Ca²⁺ currents are assumed to be a candidate ionic mechanism of theta rhythm genesis (24), analogous to the role of T-type channels in the generation of oscillations in the reticular nucleus of the thalamus (25). Nevertheless the involvement of T-type Ca²⁺ channels in hippocampal theta rhythms or exploratory behavior has not been examined. Here, we analyzed global KO mice and mice with MS-specific inactivation of the Ca_v3.1 gene encoding T-type Ca²⁺ channels, focusing on finding the neural mechanism that control the exploratory behaviors. Using a combination of tools, we provide evidence that object and open field exploratory behaviors are processed differently in the brain. Furthermore, Ca_v3.1 T-type Ca²⁺ channels in the septo-hippocampal GABAergic projection neurons are critically involved in controlling object exploration through modulating hippocampal type 2 theta rhythm.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

In cellulo phosphorylation induces pharmacological reprogramming of maurocalcin,a cell-penetrating venom peptide

The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr²⁶ belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr²⁶ phosphorylation leads to charge neutralization of Arg²⁴, a residue crucial for MCa agonist activity. The functional effect of Thr²⁶ phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs.Animal venoms represent biolibraries that are natural sources for the discovery of a growing number of peptides with important pharmacological properties. These peptides have been instrumental in discriminating different subtypes of ion channels and membrane receptors (1–3). Several of them are now approved by the US Food and Administration for the treatment of various human pathological conditions (4). Maurocalcin (MCa) is a 33-amino acid peptide that belongs to the family of the calcin toxins characterized by their folding forming an inhibitor cystine knot motif (5, 6). After its initial purification from the venom of the scorpion Scorpio maurus palmatus, MCa was obtained as a synthetic peptide that exhibits structural and pharmacological properties identical to those of the natural peptide (7). Extensive characterization of MCa identified it as one of the most potent effectors of ryanodine receptor type 1 (RyR1) (8, 9). RyR1 is one of the three isoforms that make up the RyR intracellular calcium channels family. It was first identified in skeletal muscles (10) but is also broadly expressed in many other tissues such as pre- and postsynaptic sites within neurons of the brain (11). RyR calcium channels regulate Ca²⁺ release from intracellular stores, and therefore represent crucial players of a number of physiological and toxicological processes, from muscle contraction to gene expression (12). As a consequence, silencing of the gene encoding RyR1 is lethal at birth (13). Despite extensive studies of the RyR biophysical properties, only a few molecules specifically acting on these calcium channels have been identified. The only specific inhibitors of RyR1 known so far are ryanodine at high concentration (14), although this compound also exhibits agonist properties at low doses (15, 16), and dantrolene. Ruthenium red lacks selectivity and cell permeability. On the agonist side, MCa is a very informative pharmacologic tool to investigate RyR properties because of its nanomolar potency and novel ability to stabilize long-lived subconductances of the channel. Accordingly, MCa increases specific binding of [³H]-ryanodine to RyR1 with an apparent affinity in the range of 10 to 50 nM (9). This effect results from the ability of MCa to convert RyR1 low-affinity ryanodine binding sites into high-affinity sites, increase RyR1 sensitivity to activating low Ca²⁺ concentrations (µM), and decrease RyR1 sensitivity to inhibiting high Ca²⁺ concentrations (mM), with the two latest effects probably requiring an allosteric mechanism. MCa binding onto RyR1 also induces Ca²⁺ release from sarcoplasmic reticulum vesicles (8). These effects correlate with the induction by MCa of long-lasting subconductance open transitions of purified RyR1 channels reconstituted in artificial lipid bilayers (7, 17, 18). This positive allosteric activity of MCa is the consequence of its interaction with a cytoplasmic RyR1 domain that directly controls the gating of this calcium channel (19). Production of MCa analogs after alanine scanning helped identifying critical residues involved in the positive allosteric activity of the peptide (9, 20). The pharmacophore of MCa was restricted to a few basic amino acid residues, among which Arg²⁴ was the most important for activity (9). Despite these extensive studies, none of the mutations converted MCa into RyR1 antagonist peptide, but mostly lowered affinity or inhibited agonist effect.It is highly unusual that a venom peptide targets an intracellular receptor. Several reports indicate that MCa binds cell surface glycosaminoglycans (21) and membrane lipids (20), and that it rapidly translocates into cells using mechanisms akin to cell-penetrating peptides (CPPs) (22). As a matter of fact, MCa and CPPs share interesting structural features, such as the presence of a strong dipole moment characterized by a heavily basic face opposing a far less charged one (23–25), with the latter being also quite hydrophobic. Similar to a CPP, MCa proved to be efficient in the transport of several types of cargoes ranging from drugs to peptides, proteins, and nanoparticles (22, 26–32). MCa is the first natural CPP, to our knowledge, emerging from venom sources and whose cytoplasm delivery and activity could be straightforwardly examined through the activation of RyR channels (33). It is therefore conceivable that MCa may undergo posttranslational modifications by target cell enzymes on delivery into the cytoplasm.In this report, we show that MCa is a substrate of PKA in vitro, leading to the phosphorylation of Thr²⁶. We also demonstrate that MCa phosphorylation happens in cellulo after translocation. Phosphorylation of MCa leads to a fine peptide structural modification responsible for its complete pharmacological reprogramming, thereby converting MCa into a RyR1 antagonist. We thus establish for the first time, to our knowledge, that the pharmacological properties of a small cell-penetrating toxin peptide can be dramatically modified by posttranslational modification, opening innovative approaches to develop negative allosteric modulators of the RyR1 channel, with MCa P-Thr²⁶ as a promising lead.     

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na⁺-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na⁺/leucine transporter LeuT, our results suggest that Na⁺ binding at the conserved second Na⁺ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.Secondary active transporters harness the energy of ion gradients to power the uphill movement of solutes across membranes. Mitchell (1) and others (2, 3) proposed and elaborated “alternating access” mechanisms wherein the transporter transitions between two conformational states that alternately expose the substrate binding site to the two sides of the membrane. The LeuT class of ion-coupled symporters consists of functionally distinct transporters that share a conserved scaffold of two sets of five transmembrane helices related by twofold symmetry around an axis nearly parallel to the membrane (4). Ions and substrates are bound near the middle of the membrane stabilized by electrostatic interactions with unwound regions of transmembrane helix (TM) 1 and often TM6 (4). The recurrence of this fold in transporters that play critical roles in fundamental physiological processes (5, 6) has spurred intense interest in defining the principles of alternating access.Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7–11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4, 7, 9–12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outward-facing, or occluded on the basis of the accessibility of the substrate binding site (7–11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).Mhp1, an Na⁺-coupled symporter of benzyl-hydantoin (BH) from Microbacterium liquefaciens, was the first LeuT-fold member to be characterized by crystal structures purported to represent outward-facing, inward-facing, and outward-facing/occluded conformations of an alternating access cycle (8, 15). In these structures, solvent access to ligand-binding sites is defined by the relative orientation between a 4-helix bundle motif and a 4-helix scaffold motif (8). In Mhp1, alternating access between inward- and outward-facing conformations, was predicted from a computational analysis based on the inverted repeat symmetry of the LeuT fold and is referred to as the rocking-bundle model (16). The conservation of the inverted symmetry prompted proposal of the rocking-bundle mechanism as a general model for LeuT-fold transporters (16). Subsequent crystal structures of other LeuT-fold transporters (7, 9, 10) tempered this prediction because the diversity of the structural rearrangements implicit in these structures is seemingly inconsistent with a conserved conformational cycle.Another outstanding question pertains to the ion-coupling mechanism and the driving force of conformational changes. The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na⁺ ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) simulations (20, 21) and electron paramagnetic resonance (EPR) analysis (13, 22) of LeuT demonstrated that Na⁺ binding favors an outward-facing conformation although it is unclear which Na⁺ site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na⁺ in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.Here, we used site-directed spin labeling (SDSL) (24) and double electron-electron resonance (DEER) spectroscopy (25) to elucidate the conformational changes underlying alternating access in Mhp1 and define the role of ion and substrate binding in driving transition between conformations. This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13, 26–32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra- and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward- and outward-facing conformations. A major finding is that this transition is driven by substrate but not Na⁺ binding. Although the amplitudes of the observed distance changes are in overall agreement with the rocking-bundle model deduced from the crystal structures of Mhp1 (8, 15) and predicted computationally (16), we present evidence that relative movement of bundle and scaffold deviate from strict rigid body. Comparative analysis of LeuT and Mhp1 alternating access reveal how the conserved LeuT fold harnesses the energy of the Na⁺ gradient through two distinct coupling mechanisms and supports divergent conformational cycles to effect substrate binding and release.     

Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane

Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin’s association with calcium (Ca²⁺) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca²⁺] or blocking L-type Ca²⁺ channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca²⁺ signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.Dysferlinopathies are degenerative myopathies secondary to mutations in the gene encoding the protein dysferlin. These myopathies, most commonly limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are independent of motor neuron activation (1), indicating that they are myogenic in origin. Dysferlin is a 230-kDa protein composed of seven C2 domains with homology to synaptotagmin (2, 3) and a single transmembrane domain near its C terminus (4, 5). The complexity of dysferlin’s potential role in muscle is highlighted by the number of its purported functions, including membrane repair (2, 3), vesicle fusion (4), microtubule regulation (5, 6), cell adhesion (7, 8), and intercellular signaling (9). Understanding the contributions of dysferlin to the maintenance of normal skeletal muscle function is critical for the development of appropriate therapies for patients diagnosed with LGMD2B and MM.Recently, we demonstrated the localization of dysferlin at the A-I junction in mature muscle fibers (10). These results agree with earlier reports associating dysferlin with the dihydropyridine receptor (DHPR, L-type Ca²⁺ channel), Ahnak, caveolin 3, and several other proteins involved in Ca²⁺-based signaling and the function of transverse (t-) tubules (11–14). Consistent with this localization and the potential for a functional role in this specialized compartment, dysferlin-deficient murine muscle demonstrates altered transverse tubule (t-tubule) structure (15) as well as increased oxidative stress (16, 17), inflammation, and necrosis (18–20) after injury.Here we demonstrate that dysferlin is enriched in the t-tubule membrane, where it contributes to the maintenance of the t-tubule and Ca²⁺ homeostasis. We show that, although the structure and function of dysferlin-deficient t-tubules are normal at rest, they are more readily disrupted following experimental injury and are protected by reducing extracellular [Ca²⁺] or blocking L-type Ca²⁺ channels with diltiazem. We also demonstrate that treatment of dysferlin-deficient mice with diltiazem significantly improves their recovery from injuries induced by eccentric contractions. These findings support a role for dysferlin in stabilizing the t-tubules of skeletal muscle subjected to stress and suggest that diltiazem treatment may represent a viable therapeutic option for LGMD2B and MM patients.     

Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700

L-type calcium (Ca²⁺) currents conducted by voltage-gated Ca²⁺ channel Ca_V1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca_V1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca²⁺ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the Ca_V1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca_V1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca²⁺ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.Upon membrane depolarization, Ca_V1.2 channels conduct L-type calcium (Ca²⁺) current into cardiomyocytes and initiate excitation–contraction coupling (1, 2). Ca²⁺ influx through Cav1.2 channels activates Ca²⁺ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation–contraction coupling, Ca²⁺ influx via Ca_V1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca_V1.2 channel (1, 3). Phosphorylation of the Ca_V1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca_V1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4–6). Under those pathological conditions, responsiveness of Ca_V1.2 channel activity to β-adrenergic receptors and PKA activation is severely blunted, resulting in diminished contractile reserve and impaired fight-or-flight response (6, 7). Enormous effort has been devoted to understanding how β-adrenergic regulation of the Ca_V1.2 channel is achieved, but the exact molecular mechanisms remain unresolved.Ca_V1.2 channels contain multiple subunits, including a pore-forming α₁1.2 subunit (also designated α_1C), β and α₂δ subunits that modulate expression of Ca_V1.2 at the cell surface, and possibly γ subunits (8). The closely related Ca_V1.1 and Ca_V1.2 channels in skeletal and cardiac muscle, respectively, are both proteolytically processed near the center of their large C-terminal domains (9, 10), and the distal C terminus (dCT) remains associated noncovalently with the proximal C terminus (pCT) and serves as a potent autoinhibitor (11, 12). Regulation of Ca_V1.2 channels by PKA was reconstituted in nonmuscle cells with a dynamic range of threefold to fourfold similar to native cardiomyocytes by building the autoinhibitory Ca_V1.2 complex through cotransfection of each of its components (13). Successful reconstitution required an A Kinase Anchoring Protein (AKAP), which recruits PKA to the dCT (13–15). Deletion of the dCT in vivo results in loss of regulation of the L-type Ca²⁺ current by the β-adrenergic pathway and embryonic death from heart failure (16, 17). These results suggest that the autoinhibited Ca_V1.2 signaling complex serves as the substrate for β-adrenergic regulation, and disruption of this complex leads to heart failure.PKA is responsible for phosphorylation of the Ca_V1.2 channel in response to β-adrenergic stimulation in cardiac myocytes (18–22). Although multiple PKA sites have been identified in α₁ subunits by in vitro phosphorylation (10, 23), none of these sites is required for regulation of Ca_V1.2 channels in vivo. For example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10, 24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca_V1.2 channel activity in transfected cells or cardiomyocytes (13, 25, 26). Two sites in the C terminus of the skeletal muscle Ca_V1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27). These sites are conserved in cardiac Ca_V1.2 channels as S1700 and T1704, and phosphoproteomics analysis revealed β-adrenergic–stimulated phosphorylation of S1700 by PKA (28). S1700 and T1704 reside at the interface between the pCT and dCT. In studies of the Ca_V1.2 signaling complex reconstituted in nonmuscle cells, phosphorylation of both sites was required for normal basal channel activity, whereas only S1700 was essential for PKA stimulation (13). Mutation of S1700 and T1704 to Ala in STAA mice reduced basal activity and Ca_V1.2 channel regulation by the β-adrenergic pathway in cardiomyocytes (29). To further dissect the contribution of S1700, we studied a mutant mouse line expressing Ca_V1.2 channel with the S1700A mutation (SA mice). Our results demonstrate that this single phosphorylation site is required for normal regulation of Ca_V1.2 channels, contraction of cardiac myocytes, exercise capacity, and cardiac homeostasis.     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions,regulates Ca2+ dynamics in T cells

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca²⁺ entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca²⁺ sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER–PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER–PM junctions to regulate Ca²⁺ signaling. Silencing or genetic manipulation of JP4 decreased ER Ca²⁺ content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca²⁺-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate–JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca²⁺ homeostasis and mediate SOCE in T cells.The endoplasmic reticulum (ER)–plasma membrane (PM) junctions are ubiquitous structures essential for intermembrane communications (1–3). These junctions play an important role in lipid transfer and regulation of Ca²⁺ dynamics, including ER Ca²⁺ homeostasis and Ca²⁺ entry after receptor stimulation (1, 4). Four major categories of components of the ER–PM junctions have been identified so far: (i) dyad/triad junctional proteins in the heart and skeletal muscle (e.g., junctophilins and junctin), (ii) ER-resident vesicle-associated membrane protein-associated proteins (VAPs) that form the lipid transfer machinery by interacting with phospholipid-binding proteins, (iii) extended synaptogamin-like proteins (E-Syts) that tether membranes, and (iv) the Orai1–stromal interaction molecule 1 (STIM1) complex that forms the primary Ca²⁺ channel in T cells, the Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Among these proteins, the dyad/triad junctional proteins and the Orai1–STIM1 complex are known to play a crucial role in Ca²⁺ dynamics, including excitation–contraction coupling in muscle and store-operated Ca²⁺ entry (SOCE) in immune cells, respectively (2, 5).Stimulation of T-cell receptors (TCRs) triggers activation of SOCE primarily mediated by the PM-resident Orai1 channels and ER-resident STIM1 protein that senses ER Ca²⁺ concentration (6–11). Upon store depletion, STIM1 translocates and interacts with Orai1 at the preformed ER–PM junctions (12, 13). STIM1 uses two major mechanisms to translocate into the ER–PM junctions: by interactions with phosphatidylinositol-4,5-bisphosphate (PIP₂) in the PM via its C-terminal polybasic residues and by interaction with Orai1 or the ER-resident junctate proteins (14, 15). Recently, septin filaments were shown to play a role in PIP₂ enrichment at the ER–PM junctions before STIM1 recruitment (16). Subsequently, membrane-tethering VAP and E-Syt proteins were shown to be important for PIP₂ replenishment after store depletion (17). The importance of protein interaction in STIM1 recruitment was demonstrated by a STIM1ΔK mutant truncated in its C-terminal polybasic domain. Interaction with Orai1 or junctate facilitated recruitment of this PIP₂ binding-deficient mutant into the junctions (15, 18, 19). It was thought that the roles of dyad/triad junctional proteins are limited to muscle cells. However, identification of junctate as a STIM1-interacting partner implied that some components (or homologs) of ER–PM junctions in excitable cells may be shared in immune cells.The junctophilin family consists of four genes (JP1, JP2, JP3, and JP4) that are expressed in a tissue-specific manner and are known to form ER–PM junctions in excitable cells (20, 21). Junctophilins contain eight repeats of the membrane occupation and recognition nexus (MORN) motifs that bind to phospholipids in the N terminus and a C-terminal ER membrane-spanning transmembrane segment (20, 22). In this study, we observed expression of JP4 in both human and mouse T cells, which was further enhanced by TCR stimulation. Depletion or deficiency of JP4 reduced ER Ca²⁺ content, SOCE, and activation of the nuclear factor of activated T cells (NFAT) and ERK mitogen-activated protein kinase (MAPK) pathways. Mechanistically, JP4 depletion reduced accumulation of STIM1 at the junctions without affecting the number and length of the ER–PM junctions. We observed a direct interaction between the cytoplasmic regions of JP4 and STIM1, and, correspondingly, overexpression of the STIM1-interacting JP4 fragment had a dominant negative effect on SOCE. Finally, we identified a protein complex consisting of JP4 and junctate at the ER–PM junctions, which may have a synergistic effect in recruiting STIM1 to the junctions. Therefore, our studies identify a PIP₂-independent, but protein interaction-mediated, mechanism by which the junctate–JP4 complex recruits STIM1 into the ER–PM junctions to maintain ER Ca²⁺ homeostasis and activate SOCE in T cells.     

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca²⁺ release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca²⁺ on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [³H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G^+/−) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G^+/− hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca²⁺ release during systole, gradually overloading the sarcoplasmic reticulum with Ca²⁺. The resultant Ca²⁺ overload then randomly caused bursts of prolonged Ca²⁺ release, activating electrogenic Na⁺-Ca²⁺ exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.In the heart, ryanodine receptor (RyR2) channels release massive amounts of Ca²⁺ from the sarcoplasmic reticulum (SR) in response to membrane depolarization, in turn modulating cardiac excitability and triggering ventricular contractions (1, 2). In their intracellular milieu, RyR2 channels are regulated by a variety of cytosolic and luminal factors so that their output signal (i.e., Ca²⁺) finely grades cardiac contractions (3). However, RyR2 channels operate within a limited margin of safety because conditions that demand higher RyR2 activity (such as sympathetic stimulation) also increase the vulnerability of the heart to life-threatening arrhythmias (4), and this risk is higher in hearts harboring mutant RyR2 channels. Indeed, point mutations in RYR2, the gene encoding for the cardiac RyR channel, are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) (5), a highly arrhythmogenic syndrome triggered by sympathetic stimulation that may lead to sudden cardiac death, especially in children and young adults (6).To date, delayed afterdepolarizations (DADs) triggered by spontaneous Ca²⁺ release stand as the most accepted cellular mechanism to explain cardiac arrhythmias in CPVT. In this scheme, RyR2 channels destabilized by gain-of-function mutations release Ca²⁺ during diastole, generating a depolarizing transient inward current (I_ti) as the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) extrudes the released Ca²⁺. This electrogenic inward current then causes DADs, which, if sufficiently large, reach the threshold to initiate untimely action potentials (APs) and generate triggered activity (6–8). Hence, hyperactive RyR2 channels eager to release Ca²⁺ on their own appear as essential components of this arrhythmogenic scheme. In fact, most RyR2-linked CPVT mutations characterized to date produce hyperactive RyR2 channels (9–12). This scheme therefore appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations (13).How do hypoactive RyR2 channels trigger lethal arrhythmias? Here we studied the RyR2-A4860G mutation, which was initially detected in a young girl presenting idiopathic catecholaminergic ventricular fibrillation (VF) (14). When expressed in HEK293 cells, recombinant RyR2-A4860G channels displayed a dramatic depression of activity, manifested mainly as a loss of luminal Ca²⁺ sensitivity (13). However, this in vitro characterization was insufficient to elucidate the mechanisms by which these hypoactive channels generate cellular substrates favorable for cardiac arrhythmias. We thus generated a mouse model of CPVT harboring the RyR2-A4860G mutation. Inbreeding of mice heterozygous for the mutation (RyR2-A4860G^+/−) yields only WT and heterozygous mice, indicating that the mutation is too strong to be harbored in the two RYR2 alleles. Ventricular myocytes from RyR2-A4860G^+/− mice have constitutively lower Ca²⁺ release than WT littermates, and undergo apparently random episodes of prolonged systolic Ca²⁺ release upon β-adrenergic stimulation, giving rise to early afterdepolarizations (EADs). Thus, this unique RYR2 mutation reveals novel pathways whereby RyR2 channels engage sarcolemmal currents to trigger VF. Although exposed in the setting of CPVT, this mechanism may be extended to a variety of settings, including heart failure, atrial fibrillation, and other cardiomyopathies in which RyR2 down-regulation and posttranslational modifications depress RyR2 function.