STIM1–Ca2+ signaling modulates automaticity of the mouse sinoatrial node

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca²⁺ release from internal Ca²⁺ stores (Ca²⁺ clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca²⁺ entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca²⁺ stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca²⁺ current, a reduction in L-type Ca²⁺ current, and enhancing the activities of Na⁺/Ca²⁺ exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca²⁺ dynamics in SANCs that links SR Ca²⁺ store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.Sinus rhythm of the heart is set by specialized cardiomyocytes located in the sinoatrial node (SAN). These cardiomyocytes (SANCs) lack a resting membrane potential but generate a sinus impulse after spontaneous diastolic depolarization triggers an action potential (AP). Automaticity is achieved in the SANCs by the simultaneous activation of diastolic currents during membrane depolarization and the spontaneous release of Ca²⁺ from internal stores (1–3). Several recent studies show that maintenance of sarcoplasmic reticulum (SR) Ca²⁺ stores is critically important for SAN automaticity, as is evident from genetic studies involving patients and mice that have leaky SR Ca²⁺ stores (4). Mutations in the ryanodine receptor (RYR2) or calsequestrin genes that result in spontaneous Ca²⁺ release cause catecholamiergic polymorphic ventricular tachycardia (CPVT) (5, 6). In addition to ventricular arrhythmias, these patients also develop sinus node dysfunction and bradycardia, which frequently requires permanent pacemaker insertion. Computational studies further reinforce the idea that SAN dysfunction results from leaky RYR2-containing Ca²⁺ stores (5). These studies emphasize the importance of Ca²⁺ signaling in the automaticity of SANCs. Given the emerging role of store-operated Ca²⁺ entry (SOCE) in excitable cells, we asked here whether stromal interaction molecule 1 (STIM1) plays a major role in regulating the Ca²⁺ signaling and automaticity of SANCs.SANs are structurally and functionally heterogeneous, exhibiting differences in shape and size that correspond to differences in electrophysiological features (7). It is believed that this heterogeneity is required to establish regional zones within the SAN for impulse generation by pacemakers. Under resting conditions, clusters of SANCs serve as the dominant pacemaker by firing APs at rates faster than subsidiary pacemakers located in adjacent aspects of the SAN and atria. However, under different chronotropic conditions, as occurs with autonomic stimulation, the dominant pacemaking sites can shift to different regions within the SAN, where subsidiary pacemakers can slow or speed up the heart rate (HR) (8). Dominant pacemakers are established by differences in ion channel distribution that create regional differences in electrophysiological properties such as the maximal diastolic potential (MDP) and the rate of diastolic depolarization. In addition, local intracellular factors also contribute to pacemaking by integrating currents generated at the plasma membrane with the rhythmic release of Ca²⁺ from the SR. Given the importance of Ca²⁺ dynamics to diastolic depolarization of the SAN, we hypothesize that multiple mechanisms must be available to replenish internal Ca²⁺ stores. Refilling internal Ca²⁺ stores in SANCs is known to involve Ca²⁺ entry via voltage-gated Ca²⁺ channels (9). Here we propose that Ca²⁺ store refilling also requires SOCE.In nonexcitable cells such as lymphocytes, the molecular mechanisms underlying SOCE have been well characterized and shown to require STIM1, a single-pass endoplasmic reticulum (ER) membrane protein that serves as the sensor of SR/ER Ca²⁺ store content. When Ca²⁺ stores are depleted, STIM1 molecules in the SR/ER membrane interact with plasma membrane Ca²⁺ channels, such as Orai1, to initiate Ca²⁺ entry into the cytoplasm and consequent refilling of internal Ca²⁺ stores via SR/ER Ca²⁺ pumps. SOCE is therefore an attractive candidate to link the Ca²⁺ and membrane clocks that underlie SANC automaticity. Roles for STIM1-dependent SOCE have been suggested in the heart (10–14). Here, we show that STIM1 is selectively expressed in the SAN where it activates Ca²⁺ entry via Orai1 channels and thereby modulates the Ca²⁺ signals required for pacemaking activity of the SAN. These findings build on an emerging theme, that STIM1 is a multifunctional signaling molecule, to show that STIM1 regulates several aspects of Ca²⁺ signaling in SANCs.     

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies “sick sinus” syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Ca_v1.3 Ca²⁺ channels (Ca_v1.3^−/−) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K⁺ channel (I_KACh) could rescue SSS and heart block in Ca_v1.3^−/− mice. We found that genetic inactivation of I_KACh abolished SSS symptoms in Ca_v1.3^−/− mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of I_KACh either in Ca_v1.3^−/− mice or following selective inhibition of Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current in vivo. Ablation of I_KACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from I_KACh suppression achieved by gene therapy or selective pharmacological inhibition.Pacemaker activity of the sinoatrial node (SAN) controls heart rate under physiological conditions. Abnormal generation of SAN automaticity underlies “sick sinus” syndrome (SSS), a pathological condition manifested when heart rate is not sufficient to meet the physiological requirements of the organism (1). Typical hallmarks of SSS include SAN bradycardia, chronotropic incompetence, SAN arrest, and/or exit block (1–3). SSS carries incapacitating symptoms, such as fatigue and syncope (1–3). A significant percentage of patients with SSS present also with tachycardia-bradycardia syndrome (3). SSS can also be associated with atrioventricular (AV) conduction block (heart block) (1–3). Although aging is a known intrinsic cause of SSS (4), this disease appears also in the absence of any associated cardiac pathology and displays a genetic legacy (1, 2). Heart disease or drug intake can induce acquired SSS (2). Symptomatic SSS requires the implantation of an electronic pacemaker. SSS accounts for about half of all pacemaker implantations in the United States (5, 6). The incidence of SSS has been forecasted to increase during the next 50 y, particularly in the elder population (7). Furthermore, it has been estimated that at least half of SSS patients will need to be electronically paced (7). Although pacemakers are continuously ameliorated, they remain costly and require lifelong follow-up. Moreover, the implantation of an electronic pacemaker remains difficult in pediatric patients (8). Development of alternative and complementary pharmacological or molecular therapies for SSS management could improve quality of life and limit the need for implantation of electronic pacemakers.Recently, the genetic bases of some inherited forms of SSS have been elucidated (recently reviewed in 1, 9) with the discovery of mutations in genes encoding for ion channels involved in cardiac automaticity (4, 9, 10). Notably, loss of function of L-type Ca_v1.3 Ca²⁺ channels is central in some inherited forms of SSS. For instance, loss of function in Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current causes the sinoatrial node dysfunction and deafness syndrome (SANDD) (10). Affected individuals with SANDD present with profound deafness, bradycardia, and dysfunction of AV conduction (10). Mutation in ankyrin-B causes SSS by reduced membrane targeting of Ca_v1.3 channels (11). The relevance of Ca_v1.3 channels to SSS is demonstrated also by work on the pathophysiology of congenital heart block, where down-regulation of Ca_v1.3 channels by maternal Abs causes heart block in infants (12). Additionally, recent data show that chronic iron overload induces acquired SSS via a reduction in Ca_v1.3-mediated I_Ca,L (13).In mice and humans, Ca_v1.3 channels are expressed in the SAN, atria, and the AV node but are absent in adult ventricular tissue (14, 15). Ca_v1.3-mediated I_Ca,L plays a major role in the generation of the diastolic depolarization in SAN and AV myocytes, thereby constituting important determinants of heart rate and AV conduction velocity (14, 16). The heart rate of mice lacking Ca_v1.3 channels (Ca_v1.3^−/− mice) fairly recapitulates the hallmarks of SSS and associated symptoms, including bradycardia and tachycardia-bradycardia syndrome (17, 18). In addition, severe AV dysfunction is recorded in Ca_v1.3^−/− mice to variable degrees. Typically, these mice show first- and second-degree AV block (16, 17, 19). Complete AV block with dissociated atrial and ventricular rhythms can also be observed in these animals. The phenotype of Ca_v1.3^−/− mice thus constitutes a unique model for developing new therapeutic strategies against SSS (10).The muscarinic-gated K⁺ channel (I_KACh) is involved in the negative chronotropic effect of the parasympathetic nervous system on heart rate (20, 21). Two subunits of the G-protein activated inwardly rectifying K⁺ channels (GIRK1 and GIRK4) of the GIRK/Kir3 subfamily assemble as heterotetramers to form cardiac I_KACh channels (22). Indeed, both Girk1^−/− and Girk4^−/− mice lack cardiac I_KACh (20, 21, 23). We recently showed that silencing of the hyperpolarization-activated current “funny” (I_f) channel in mice induces a complex arrhythmic profile that can be rescued by concurrent genetic ablation of Girk4 (24). In this study, we tested the effects of genetic ablation and pharmacological inhibition of I_KACh on the Ca_v1.3^−/− mouse model of SSS. We found that Girk4 ablation or pharmacological inhibition of I_KACh rescues SSS and AV dysfunction in Ca_v1.3^−/−. Thus, our study shows that I_KACh targeting may be pursued as a therapeutic strategy for treatment of SSS and heart block.     

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.     

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.     

TRPM2 channels mediate acetaminophen-induced liver damage

Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca²⁺ homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca²⁺ concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca²⁺ rise. Here we report that the channel responsible for Ca²⁺ entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H₂O₂ or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H₂O₂. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.Acetaminophen (N-acetyl-p-aminophenol), when used at prescribed doses, is a safe analgesic and antipyretic drug (1). Its overdose, however, can be life threatening, causing severe liver and kidney damage (2–5). In Western countries, acetaminophen-induced hepatotoxicity is a leading cause of acute liver failure requiring liver transplantation (6). Due to a widespread availability of acetaminophen and potentially lethal consequences of its overdose, the mechanisms of acetaminophen hepatotoxicity have been in focus of a large number of investigations (7). Despite significant progress, the exact pathways of acetaminophen hepatotoxicity that lead to hepatocellular death are still not completely understood. It is clear, however, that acetaminophen toxicity arises from its metabolic activation (8, 9).In the liver, therapeutic doses of acetaminophen are metabolized by glucuronidation and sulfation into nontoxic compounds (1). Only a small amount of acetaminophen is converted by hepatic cytochrome P450 (CYP)-dependent mixed function oxidases to the reactive intermediate metabolite N-acetyl-parabenzo-quinoneimine (NAPQI). The NAPQI generated by a therapeutic dose of acetaminophen is rapidly metabolized to nontoxic products by conjugation with glutathione (GSH) (1, 10). With large doses of acetaminophen, however, hepatic GSH becomes depleted resulting in the accumulation of toxic amounts of NAPQI. Covalent binding of NAPQI to cellular proteins has previously been considered the main cause of liver cell death under these circumstances. Indeed it has been shown that covalent binding precedes hepatocellular death, and treatments that prevent covalent binding also prevent liver necrosis (11). More recently, however, it has been suggested that, by itself, the covalent binding of NAPQI is not sufficient to induce apoptosis or necrosis. The toxic signal produced by covalent binding undergoes further amplification through the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), deregulation of Ca²⁺ homeostasis, and increased intracellular Ca²⁺, causing oxidant stress in mitochondria and inducing the mitochondrial membrane permeability transition (12, 13). Although widely acknowledged, the role of Ca²⁺ in acetaminophen toxicity is poorly understood and has not been thoroughly investigated. Nonselective Ca²⁺ channels blockers chlorpromazine and verapamil have been shown to attenuate liver injury in mice (14, 15), however, the mechanism of their protective properties in acetaminophen overdose is not clear, and it is not known whether it involves any Ca²⁺-permeable channels on the plasma membrane of hepatocytes.The only Ca²⁺-selective channel that has been clearly identified in hepatocytes so far is the Ca²⁺ release-activated Ca²⁺ channel activated by the depletion of intracellular Ca²⁺ stores downstream of phospholipase C_β and phospholipase C_γ signaling (16, 17). In addition, a number of Ca²⁺-permeable nonselective cation channels with no clearly defined functions and mostly from the TRP family of channels have been shown to be present in hepatocytes and liver cells (18, 19). One of these channels, Transient Receptor Potential Melanostatine 2 (TRPM2), whose presence in the liver has only been demonstrated on an mRNA level (19), is activated in response to oxidative stress and, potentially, can be involved in acetaminophen-induced Ca²⁺ rise in hepatocytes.     

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.     

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca²⁺ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca²⁺-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca²⁺ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca²⁺ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca²⁺ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca²⁺, intracellular Ca²⁺ buffering, and membrane potential, by their common effect on intracellular free Ca²⁺.Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca²⁺ entry through the channel itself (6–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca²⁺, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca²⁺ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca²⁺-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca²⁺ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20–23) compared with the piezo-driven glass rod (19, 24).     

Mitochondrial calcium uniporter regulator 1 (MCUR1) regulates the calcium threshold for the mitochondrial permeability transition

During the mitochondrial permeability transition, a large channel in the inner mitochondrial membrane opens, leading to the loss of multiple mitochondrial solutes and cell death. Key triggers include excessive reactive oxygen species and mitochondrial calcium overload, factors implicated in neuronal and cardiac pathophysiology. Examining the differential behavior of mitochondrial Ca²⁺ overload in Drosophila versus human cells allowed us to identify a gene, MCUR1, which, when expressed in Drosophila cells, conferred permeability transition sensitive to electrophoretic Ca²⁺ uptake. Conversely, inhibiting MCUR1 in mammalian cells increased the Ca²⁺ threshold for inducing permeability transition. The effect was specific to the permeability transition induced by Ca²⁺, and such resistance to overload translated into improved cell survival. Thus, MCUR1 expression regulates the Ca²⁺ threshold required for permeability transition.The mitochondrial permeability transition (MPT) pore is large, and its opening collapses the mitochondrial membrane potential (ΔΨ), depleting the matrix of solutes <1.5 kDa. The osmotic imbalance swells and disrupts mitochondria, leading to cell death. The molecular structure of the MPT pore is unknown, although cyclophilin D [peptidyl-prolyl isomerase F (PPIF)], the ADP/ATP translocase, the F1-FO-ATP synthase, and spastic paraplegia 7 are key for its function (1–5).Key triggers for the MPT include oxidative damage and Ca²⁺ overload. Reactive oxygen species attack a cysteine residue in mammalian PPIF (6, 7), but how Ca²⁺ overload activates the pore is unknown. Elimination of the known regulators typically inhibits the sensitivity of the MPT globally, not favoring any particular trigger (8–10). Because Ca²⁺ overload promotes cell death in excitable cells, targeting this pathway selectively may prove beneficial.To discover novel regulators specific to mitochondrial Ca²⁺ overload, we studied MPT in Drosophila S2R+ cells, a system where screens have identified molecules involved in Ca²⁺ transport (11–13). We found that mitochondria within these cells were resistant to Ca²⁺ overload (14) but did possess an MPT. Moreover, we identified a mammalian gene, mitochondrial calcium uniporter regulator 1 (MCUR1), with no known Drosophila homolog, which is able to alter the MPT Ca²⁺ threshold. Inhibiting this gene confers resistance from cell death mediated by mitochondrial Ca²⁺ overload.     

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.     

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl? channels

Ca²⁺-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca²⁺ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP)—we discovered that Ca²⁺-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca²⁺-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM₁₂₃₄. These effects are Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca²⁺-activated chloride channels.Calcium (Ca²⁺)-activated chloride (Cl^–) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3–5), smooth muscle contraction (6), olfactory transduction (7, 8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).Three laboratories independently identified the transmembrane protein TMEM16A as the molecular component of a CaCC (12–14). TMEM16A belongs to a protein family with 10 members encoded by distinct genes (15–18). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2, 12–14, 19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20–22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2, 19). Human TMEM16A has four alternatively spliced segments (a–d), differential inclusion of which modify voltage and Ca²⁺ sensitivity of resultant channel splice variants (24).CaCCs are highly sensitive to intracellular [Ca²⁺], displaying graded increases in Cl⁻ current (I_Cl) amplitude as [Ca²⁺]_i is raised from resting levels (∼100 nM) to the 1- to 2-μM range. In some cases, high [Ca²⁺]_i (>10 μM) leads to decreased I_Cl amplitude (inactivation) (25–27). The Ca²⁺ sensor(s) for Ca²⁺-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is unknown. There are two possible nonexclusive mechanisms: (i) direct Ca²⁺ binding to the channel or (ii) Ca²⁺ binding through a separate Ca²⁺-sensing protein. The TMEM16A sequence does not reveal any canonical Ca²⁺-binding EF hand motifs (14, 16, 17). A sequence in the first intracellular loop of TMEM16A resembling the “Ca²⁺ bowl” in large conductance Ca²⁺-activated K⁺ (BK) channels was disqualified by mutagenesis as the Ca²⁺ sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca²⁺ sensitivity of TMEM16A (29, 30).Some reports have suggested involvement of calmodulin (CaM) in distinct aspects of Ca²⁺-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segment b (31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is contradicted by the robust CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segment b (2, 24). Recently, Ca²⁺–CaM was found to bind TMEM16A(ac) in a Ca²⁺-dependent manner and result in an increased permeability of the channel to HCO₃⁻ (32). Deleting the Ca²⁺–CaM binding site did not affect CDA of TMEM16A(ac). Ca²⁺–CaM regulation of TMEM16A HCO₃⁻ permeability conforms to a traditional signaling mode where Ca²⁺ binds to freely diffusing CaM to form a Ca²⁺–CaM complex that then interacts with a target protein.There are several examples of an alternative mode of CaM signaling in which Ca²⁺-free CaM (apoCaM) is preassociated with target proteins under resting [Ca²⁺]_i conditions and acts as a resident Ca²⁺ sensor to regulate function of the host protein in response to increased [Ca²⁺]_i (33). This mode of CaM signaling is used as the activating mechanism for small conductance K⁺ channels (34) and Ca²⁺-dependent regulation of high voltage-gated Ca²⁺ (Ca_V1 and Ca_V2) channels (35, 36). A distinguishing feature of this mode of CaM signaling is that it is impervious to pharmacological inhibitors of Ca²⁺–CaM, but can be eliminated in dominant negative fashion by a CaM mutant, CaM₁₂₃₄, in which all four EF hands have been mutated so they no longer bind Ca²⁺ (34, 36). Whether apocalmodulin preassociates with TMEM16A/TMEM16B channel complexes and participates in Ca²⁺-dependent regulation of these channels is controversial (30, 37, 38).Using a recently developed bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP) (39)—we discovered that apoCaM is functionally preassociated with TMEM16A/16B channel complexes. Whereas the resident apoCaM is not necessary for CDA, it does mediate two distinct Ca²⁺-dependent processes in TMEM16A. First, it causes a leftward shift in the Ca²⁺-dependent activation curve at [Ca²⁺]_i ≤ 1 μM, an effect we term Ca²⁺-dependent “sensitization” of activation (CDSA). Second, it is the Ca²⁺ sensor for CDI observed at [Ca²⁺]_i > 10 μM. The two opposite effects are independently mediated by the two lobes of preassociated apoCaM— Ca²⁺ occupancy of the N lobe leads to CDI, whereas the C lobe mediates CDSA. Alternative splicing of TMEM16A provides a mechanism for regulating apoCaM binding and signaling. TMEM16A splice variants lacking segment a lost apoCaM binding altogether, eliminating both CDSA and CDI, whereas variants specifically lacking segment b were selectively deficient in CDI. Finally, TMEM16A variants defective in apoCaM binding displayed dramatically decreased trafficking to the cell surface.     

A role for the mevalonate pathway in early plant symbiotic signaling

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca²⁺ concentration (Ca²⁺ spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca²⁺ spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume–rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca²⁺ spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca²⁺ spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca²⁺ spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca²⁺ spiking in this heterologous system.The mevalonate (MVA) pathway controls the biosynthesis of hundreds of isoprenoids (sterols, carotenoids, prenyl side chains, etc.) in eukaryotes. These isoprenoids contribute to membrane integrity and development, among many other functions (1). Here, we report that the MVA pathway is also necessary for the earliest responses of plants to symbiotic signals produced by nitrogen-fixing rhizobia and arbuscular mycorrhizal (AM) fungi. These two types of endosymbiotic associations require a common set of genes in host plants to allow successful bacterial and fungal colonization. The molecular mechanisms controlling the establishment of the legume–rhizobium symbiosis have been extensively studied in model legumes such as Medicago truncatula and Lotus japonicus (2, 3). Rhizobia secrete lipochitooligosaccharides (LCOs) known as nodulation (Nod) factors, which are perceived by LysM-type receptor kinases, such as Nod factor perception (NFP) and LYK3 in M. truncatula and are required for both rhizobial infection and nodule organogenesis (4, 5). Similarly, AM fungi release signal molecules, so-called Myc factors, which are likely perceived by other LysM-type receptor kinases in both legumes as well as nonleguminous plants (6–8). The perception of Nod and Myc factors initiates early symbiotic responses in host plants through the activation of the receptor-like kinase Does not Make Infections 2 (DMI2), which is believed to act as a coreceptor (9). Although these signaling components reside on the plasma membrane (10, 11), the perception of symbiotic signals triggers sustained oscillations in Ca²⁺ concentration both within the nucleus (nuclear Ca²⁺ spiking) and around the nucleus (perinuclear Ca²⁺ spiking) (12, 13). As a result, the generation of second messengers transducing the signals from the plasma membrane to the nuclear envelope has long been hypothesized (12–19).Elegant mathematical models have been developed to explain the mechanism of nuclear Ca²⁺ spiking and the primary role of DMI1 (a nuclear envelope-localized cation channel) in its initiation and maintenance (13, 18, 20–22). Downstream decoding of Ca²⁺ spiking involves the nuclear Ca²⁺/calmodulin-dependent protein kinase DMI3 (23). In M. truncatula, DMI1, DMI2, and DMI3 are essential components of the common symbiosis pathway that is required for establishing both root nodulation and the AM symbiosis, as the respective mutants are defective for both symbioses (2). DMI1 is thus viewed as the first known target of the unidentified second messenger(s) transducing signals from the plasma membrane to the nucleus.In a previous study, a yeast two-hybrid screen identified a 3-hydroxy 3-methylglutaryl CoA reductase 1 (HMGR1) as strongly interacting with DMI2 (24). HMGRs are well-known regulatory enzymes of the MVA pathway in plants and animals, catalyzing the conversion of HMG-CoA into MVA. Furthermore, the pharmacological inhibition of HMGRs by statin drugs led to decreased nodulation (24). More specifically, silencing HMGR1 by RNA interference (RNAi) resulted in a drastic reduction in root infection and nodule development (24). However, despite these findings, the precise role of HMGR1 and MVA in symbiosis remained unclear. We now demonstrate that HMGR1 plays a key role during the initial symbiotic signaling between the host plant and both rhizobia and AM fungi. Using pharmacological, biochemical, and genetic approaches, we show that HMGR1 and the products of the MVA pathway act upstream of DMI1 in the symbiotic signaling cascade, providing the missing link between the perception of symbiotic signals at the plasma membrane and the activation of Ca²⁺ spiking in the nucleus.     

SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility

Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca²⁺ and K⁺, leading to an elevation in cytosolic Ca²⁺ critical for activation of hyperactivated swimming motility. In mice, the Ca²⁺ conductance (alkalization-activated Ca²⁺-permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K⁺ conductance (sperm pH-regulated K⁺ current, KSPER) arises from a pore-forming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca²⁺ and K⁺ conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions.Upon entry into the female reproductive tract, mammalian sperm undergo a sequence of maturational steps, collectively termed capacitation, to become competent to fertilize an egg (1, 2). Two important components of this process, thought to be shared among mammalian species, are cytosolic alkalization (3–5) and then an associated increase in cytosolic Ca²⁺ (6, 7). These events are coupled with changes in ionic fluxes in sperm membrane. Over the past 10 y, the application of patch-clamp recording to individual sperm (8) has allowed identification of ionic currents that respond to alkalization and/or Ca²⁺ (9–12). In mouse sperm, alkalization leads to activation of two sperm-specific channels, the Ca²⁺-permeable CATSPER channel (8–10, 13) and the K⁺-permeable KSPER K⁺ channel (14, 15). KSPER and CATSPER currents are also present in human sperm (16–18), although intriguingly there seem to be differences in regulation of each channel type between mice and humans (11, 12, 17).Despite the species-specific differences in the details of their regulation, KSPER and CATSPER are of central importance in sperm function and fertility in both humans and mice. In mouse sperm, KSPER and CATSPER together account for all patch-clamp measurable cation current activated by voltage and alkalization (19) and are thought to act in concert to mediate the changes in membrane cation conductance and Ca²⁺ influx that occur during the onset of capacitation (14, 20). The critical role of both KSPER and CATSPER in the mouse has been established by demonstration that genetic KO of the pore-forming subunits of each channel [KSPER (15, 21) and CATSPER (22–26)] results in infertility and the CATSPER auxiliary δ subunit is also required for fertility (27).For mouse KSPER, the pore-forming subunit is termed SLO3, encoded by the kcnu1 (slo3) gene (28). SLO3, which is exclusively expressed in testis (15, 28), is a homolog of the Ca²⁺- and voltage-activated, large-conductance (BK)-type K⁺ channel (29) and heterologously expressed SLO3 results in voltage- and alkalization-activated K⁺ currents (28). However, heterologously expressed mouse SLO3 channels differ from mouse KSPER current in important ways. Specifically, whereas pH 7 substantially activates KSPER in mouse sperm at membrane potentials (V_m) between −10 and −50 mV, activation of SLO3 channels at pH 7 is scarcely observed even at an activation potential of +100 mV (30, 31). This discrepancy raised the possibility that an additional regulatory partner of SLO3 may be present in mouse sperm. Guided by the discovery of a new type of auxiliary subunit of the BK channel (32), we recently showed that a related subunit, LRRC52 (leucine-rich-repeat-containing protein 52) is selectively expressed in sperm (33). When LRRC52 is coexpressed with SLO3, the gating range of the resulting channels at a given pH is more like that of KSPER in mouse sperm. LRRC52 protein has also been shown to be present in human sperm and, when coexpressed with human SLO3, LRRC52 results in currents with properties similar to those of human KSPER (12). To evaluate the importance of the LRRC52 subunit, we have now generated lrrc52^−/− (LRRC52 KO) mice. LRRC52 KO mice have a severe fertility deficit that is associated with a shift to more positive potentials in the KSPER current activation range in the LRRC52 KO sperm. Furthermore, measurement of the net KSPER current in sperm from two other genotypes, slo3^+/− and a slo3-eGFP, suggests that sperm reproductive capacity strongly depends on the KSPER conductance available over physiological potentials. These results confirm that LRRC52 is a critical component of the KSPER channel complex and is essential for male fertility.     

In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor

cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca²⁺ biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O₂ can evoke a tonic increase in cGMP that requires an atypical O₂-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca²⁺ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca²⁺ stimulate competing negative feedback loops that shape cGMP dynamics. Ca²⁺-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O₂, apparently by keeping in check gating of cGMP channels and limiting activation of Ca²⁺-dependent negative feedback loops. Simultaneous imaging of Ca²⁺ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O₂-evoked cGMP and Ca²⁺ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.The second messenger cyclic guanosine monophosphate (cGMP) regulates a range of physiological processes. In nervous systems, it can transduce sensory inputs (1) and modulate neuronal excitability and learning (2) and is implicated in control of mood and cognition (3). Precise regulation of cGMP levels ([cGMP]) is thought critical for these functions. This importance has prompted development of genetically encoded cGMP indicators, with the goal of visualizing cGMP dynamics with high temporal and spatial resolution (4, 5). Although these sensors have been used to image pharmacologically evoked changes in cGMP in cultured cells or tissue slices (6–10), endogenous cGMP dynamics have not been visualized and functionally dissected in vivo in any nervous system (4, 5).Local [cGMP] reflects the net activity of guanylate cyclases (GCs) that synthesize cGMP (11) and phosphodiesterases (PDEs) that degrade it (12, 13). Mammals have several families of GCs (14, 15) and eight families of cGMP PDEs (16), each with distinct regulatory properties. Different PDE types are often coexpressed, but little is known about how they work together. cGMP signaling alters cell physiology by controlling cGMP-dependent protein kinases (PKG) (17, 18), cGMP-gated channels (CNGC) (19), and cGMP-regulated PDEs (12). These cGMP effectors can also feed back to control cGMP dynamics.cGMP is a major second messenger in Caenorhabditis elegans, implicated in the function of a third of its sensory neurons, including thermosensory, olfactory, gustatory, and O₂-sensing neurons (20). Genetic and behavioral studies suggest that cGMP mediates sensory transduction in many of these neurons (21–25). Despite this pervasiveness, cGMP has not been visualized in any C. elegans cell: genetic inferences about its roles in signal transduction are untested, and we have no mechanistic insights into cGMP signaling dynamics and feedback control. Consistent with the prominence of cGMP signaling in the nematode, the C. elegans genome encodes 34 GCs (26), six PDE genes, at least one PKG (24, 27, 28), and six CNGC subunits (19, 21, 22, 29–31).Here, we use cGMP and Ca²⁺ sensors to visualize and dissect cGMP signaling dynamics in a C. elegans O₂ sensor. We image single and double mutants defective in a soluble guanylate cyclase (sGC), CNGC subunits, PDE-1, PDE-2, and PKG. Our results reveal a signaling network of interwoven checks and balances. Counterintuitively, cGMP activation of PDE-2 promotes cGMP accumulation by controlling gating of CNGC and limiting Ca²⁺-mediated negative feedback, including activation of PDE-1. We show that cGMP signal transduction is highly reliable when the same individual is stimulated repeatedly but, surprisingly, is highly variable across genetically identical, similarly reared animals. Finally, simultaneous imaging of O₂-evoked cGMP and Ca²⁺ responses suggests that cGMP dynamics can differ in distinct subcellular compartments of a C. elegans neuron, consistent with the existence of cGMP nanodomains.     

N-terminal domain of complexin independently activates calcium-triggered fusion

Complexin activates Ca²⁺-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca²⁺-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca²⁺-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca²⁺-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca²⁺-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca²⁺-triggered release.Neurotransmitter release occurs upon fusion of synaptic vesicles with the plasma membrane (1, 2). Synaptic vesicle fusion is orchestrated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fusion proteins (3, 4), in conjunction with synaptotagmin, complexin, and other synaptic proteins. The Ca²⁺-sensor synaptotagmin is essential for Ca²⁺-triggered release (5–8). Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein 25 (SNAP-25). The main isoform synaptotagmin-1 is involved in synchronous release, and forms a conserved Ca²⁺-independent interface with the ternary SNARE complex (9), along with Ca²⁺-dependent interactions with the plasma membrane, and potentially other interfaces with the SNARE complex (10). Complexin is a small cytosolic α-helical protein abundant in the presynaptic terminal (11) that interacts with the SNARE complex (12) and the membrane (13).Complexin has at least two functions: It “activates” (i.e., greatly enhances) Ca²⁺-triggered synchronous neurotransmitter release by cooperating with synaptotagmin, and it regulates spontaneous release in the presynaptic terminal (recently reviewed in refs. 14–16). The activating function of complexin is conserved across all species (mammals, Drosophila, and Caenorhabditis elegans) and different types of Ca²⁺-triggered synaptic vesicle fusion studied to date (11, 17–26). Complexin also regulates spontaneous neurotransmitter release, although this effect is less conserved among species and varies depending on experimental conditions: for example, in Drosophila, spontaneous release increases with knockout of complexin (27, 28). Likewise, knockdown of complexin in cultured cortical neurons increases spontaneous release, although knockout of complexin in mice only affects spontaneous release depending on the particular neuronal cell type (20, 23, 24, 29). Exactly how complexin can exhibit these dual effects on Ca²⁺-triggered and spontaneous synaptic vesicle fusion remains enigmatic; however, it is known that different domains of complexin play different roles in Ca²⁺-triggered and spontaneous vesicle fusion, as summarized in the following paragraphs.Here, we focus on the complexin-1 isoform (referred to as Cpx in the following). Cpx can be divided into four domains (Fig. 1A, Bottom) that are involved in different functions. The N-terminal domain (residues 1–27) of Cpx is important for activation of synchronous Ca²⁺-triggered release in murine neurons (20, 30, 31) and in isolated chromaffin cells (32). However, N-terminal truncation of Cpx in C. elegans neuromuscular junctions does not decrease Ca²⁺-triggered release, but rather increases spontaneous release (21, 22), perhaps suggesting that reduction of activation may have been masked by a simultaneous increase of spontaneous fusion in these previous experiments.Open in a separate windowFig. 1.Cpx (26–83) fragment reduces spontaneous fusion similar to wild-type Cpx. (A) Schematic diagram of the single-vesicle content mixing assay (35) (Methods) and domain diagrams of Cpx and Cpx fragments used in this figure. PM, vesicles with reconstituted syntaxin-1A and SNAP-25A that mimic the plasma membrane; SV, vesicles with reconstituted synaptobrevin-2 and synaptotagmin-1 that mimic synaptic vesicles. The bar graphs show the effects of 2 μM Cpx or Cpx fragments on the SV/PM vesicle association count during the first acquisition periods (Methods) (B), the average probability of spontaneous fusion events per second (C), the amplitude of the first 1-s time bin (probability of a fusion event in that bin) upon Ca²⁺ injection (D), and the decay rate (1/τ) of the histogram upon Ca²⁺ injection (E). The fusion probabilities and amplitudes were normalized with respect to the corresponding number of analyzed SV/PM vesicle pairs (Methods). Individual histograms are in Figs. S1 and ​andS2.S2. The error bars in B–D are SDs for multiple independent repeat experiments (20, 29, 30, 33–38). Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx for Ca²⁺-triggered release compared with wild-type neurons in rescue experiments of Cpx knockdown (23, 39).The central domain of Cpx (residues 49–70) is essential for all functions of complexins in all species studied to date, including priming (23, 24, 39, 40), inhibiting spontaneous release (18, 20–22, 35, 37, 38), and activation of Ca²⁺-triggered release (17, 18, 20, 22, 30, 31, 35, 41).The C-terminal domain binds to phospholipids (24, 42), and it is important for vesicle priming in neurons (24, 32, 43). Moreover, Cpx without the C-terminal domain does not reduce spontaneous release in neuronal cultures, but it still activates Ca²⁺-triggered release in neuronal cultures (24) and in a reconstituted system (35). The C-terminal domain is sensitive to membrane curvature, and it may thus localize Cpx to the synaptic membrane (13, 44).Structurally, in isolation, both the N- and C-terminal domains of Cpx are largely flexible, although the accessory and central domains have α-helical propensity (45). The α-helical central domain of Cpx binds to the groove between the synaptobrevin-2 and syntaxin-1A α-helices in the center of the neuronal SNARE complex (12, 46). Cpx has two conformations when bound to the ternary SNARE complex, one of which induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of Cpx (47).Cpx has been studied extensively with reconstituted systems (35, 38, 48–52). The single-vesicle fusion assay described by Lai et al. (35) qualitatively reproduced the effects of synaptotagmin-1 and Cpx in both spontaneous and Ca²⁺-triggered release that have been observed in cortical neuronal cultures (9, 35).Here, we conducted single-vesicle fusion and single-molecule membrane-binding experiments to obtain new insights into the function of the Cpx N-terminal domain. We found that the N-terminal domain can be physically separated from the accessory and central domains of Cpx and still preserve its role in activating Ca²⁺-triggered release. The N-terminal domain interacts with membranes, an interaction that is enhanced by the presence of SNARE complex. Moreover, the N-terminal domain of full-length Cpx can be functionally substituted by the fusion peptide of influenza virus hemagglutinin (HA), suggesting that similar fusion elements and principles are used in different contexts of biological membrane fusion.     

Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis

Signaling through the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel regulates critical cellular functions, including gene expression, cell growth and differentiation, and Ca²⁺ homeostasis. Loss-of-function mutations in the CRAC channel pore-forming protein ORAI1 or the Ca²⁺ sensing protein stromal interaction molecule 1 (STIM1) result in severe immune dysfunction and nonprogressive myopathy. Here, we identify gain-of-function mutations in the cytoplasmic domain of STIM1 (p.R304W) associated with thrombocytopenia, bleeding diathesis, miosis, and tubular myopathy in patients with Stormorken syndrome, and in ORAI1 (p.P245L), associated with a Stormorken-like syndrome of congenital miosis and tubular aggregate myopathy but without hematological abnormalities. Heterologous expression of STIM1 p.R304W results in constitutive activation of the CRAC channel in vitro, and spontaneous bleeding accompanied by reduced numbers of thrombocytes in zebrafish embryos, recapitulating key aspects of Stormorken syndrome. p.P245L in ORAI1 does not make a constitutively active CRAC channel, but suppresses the slow Ca²⁺-dependent inactivation of the CRAC channel, thus also functioning as a gain-of-function mutation. These data expand our understanding of the phenotypic spectrum of dysregulated CRAC channel signaling, advance our knowledge of the molecular function of the CRAC channel, and suggest new therapies aiming at attenuating store-operated Ca²⁺ entry in the treatment of patients with Stormorken syndrome and related pathologic conditions.Ca²⁺ influx in response to the depletion of intracellular Ca²⁺ stores, or store-operated Ca²⁺ entry, constitutes one of the major routes of Ca²⁺ entry in all animal cells (1). Under physiological conditions, Ca²⁺ influx is activated in response to numerous G protein-coupled receptors and receptor tyrosine kinases signaling via inositol-1,4,5-trisphosphate as a second messenger (2). Store-operated Ca²⁺ entry is mediated primarily by the Ca²⁺ release-activated Ca²⁺ (CRAC) channel (3), which consists of the pore-forming subunits ORAI1–3 (or CRAC modulators 1–3) and Ca²⁺ sensors, STIM1 and STIM2 (4–7). STIM proteins reside in the membrane of endoplasmic reticulum (ER), whereas ORAI proteins reside in the plasma membrane. STIM1 is a single transmembrane-spanning protein (8–12) that, in resting cells, exists as a dimer that binds Ca²⁺ through two EF hand-containing domains located in the ER lumen (13). Depletion of Ca²⁺ in the ER induces a series of molecular events in the conformation and localization of STIM1, initiated by the formation of higher-order oligomers, protein unfolding, and accumulation at discrete sites in the cell where the ER membrane is in close proximity to the plasma membrane (11, 13–16). In these sites, STIM1 binds to the cytosolic C and N termini of ORAI1 (17, 18), resulting in channel activation and generation of a highly Ca²⁺-selective CRAC current, or I_CRAC (3, 19, 20). I_CRAC is responsible not only for restoring cytosolic and ER Ca²⁺ concentration, thus maintaining the cell in a Ca²⁺ signaling-competent stage (1), but also for many cellular functions such as regulation of gene expression, exocytosis, proliferation, and apoptosis (1).Consistent with a fundamental role of the CRAC channel in cell signaling, loss-of-function mutations in STIM1 or ORAI1 lead to immune deficiency and nonprogressive myopathy (21–23). However, evidence that gain-of-function mutations in STIM1 and ORAI1 can affect human health is only recently starting to emerge. It was shown that mutations in the domain of STIM1 that binds Ca²⁺ (EF hand domain) in resting conditions are associated with nonsyndromic myopathy with tubular aggregates (24). Functional studies demonstrated that these mutations cause hyperactivation of the CRAC channel (24). However, it remains unknown whether myopathy with tubular aggregates is caused by the increased activity of the CRAC channel, increased activity of another Ca²⁺ channel using STIM1 as a sensor (25), or a function of STIM1 that is unrelated to Ca²⁺ signaling, as STIM1 can function independently of ORAI1 (26-28).Stormorken syndrome [Mendelian Inheritance in Man (MIM) 185070] is a rare autosomal-dominant condition with a constellation of symptoms, including congenital miosis, bleeding diathesis, thrombocytopenia, functional (or anatomical) asplenia, and proximal muscle weakness (29). Other manifestations include ichthyosis, headaches, and dyslexia (30). Patients typically display increased creatine kinase (CK) levels and histologic evidence of myopathy with tubular aggregates (30, 31). Here, we show that Stormorken syndrome is caused by an activating mutation in STIM1. We also identify a mutation in the STIM1-interacting molecule, ORAI1, in a Stormorken-like syndrome that presented with miosis and tubular myopathy. Functional analyses reveal that both mutations enhance the activity of the CRAC channel, but by different molecular mechanisms. These data expand the phenotypic spectrum of activating mutations in the CRAC channels from myopathy with tubular aggregates to miosis, bleeding diathesis, thrombocytopenia, asplenia, ichthyosis, headaches, and dyslexia.     

Pathophysiological implication of CaV3.1 T-type Ca2+ channels in trigeminal neuropathic pain

A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in Ca_V3.1 T-type Ca²⁺ channel knockout and wild-type mice. Ca_V3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in Ca_V3.1 knockout mice. Our results suggest that the presence of Ca_V3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.Since 1911, when H. Head and G. M. Holmes first addressed the relevance of the thalamus as a central pattern generator for neuropathic pain (1), many clinical studies have indicated the coexistence of pathophysiological thalamocortical activity and the occurrence of neuropathic pain. Compared with healthy controls, patients with neuropathic pain show increased low-frequency thalamocortical oscillations in magnetoencephalogram (MEG) recordings. Such low-frequency oscillations are a typical thalamocortical dysrhythmia (TCD) syndrome (2). In agreement with such MEG findings, the excess power of low-frequency oscillation was marked in local-field potential (LFP) recordings from the thalamus (3–5) and electroencephalogram (EEG) recordings from the cortex (6, 7) of patients with neuropathic pain. In addition, the presence of thalamic burst firing, which is a well-known underlying mechanism for cortical low-frequency oscillations through the thalamocortical recurrent network (8, 9), has been confirmed in patients with neuropathic pain (10–13). Results from small lesions in the posterior part of the central lateral nucleus of the medial thalamus, which reduce tonic hyperpolarization of thalamic neurons in chronic neuropathic pain patients, have also provided insight into the role of low-frequency thalamic rhythmicity in neuropathic pain. Following such interventions, a marked decrease in low-frequency EEG power was observed as well as pain relief (7, 12), indicating that alteration of thalamocortical rhythms plays a crucial role in the development and/or persistence of neuropathic pain.Trigeminal neuropathic pain (TNP) is characterized by unilateral chronic facial pain limited to one or more divisions of the trigeminal nerve. There is increasing evidence that TNP is associated with anatomical and biochemical changes in the thalamus (14–16). Moreover, patients with TNP display significant reductions in thalamic volume and neural viability (15), indicating that altered thalamic anatomy, physiology, and biochemistry may result in disturbed thalamocortical oscillatory properties.Abnormal thalamic activity has been investigated in patients with neuropathic pain (3–7, 10–13, 17, 18), including TNP (14–16). Furthermore, the potential role of thalamic burst firing in abnormally increased low-frequency oscillations has been proposed as a pathophysiological mechanism (2, 12). Because T-type Ca²⁺ channels are known to underlie thalamic burst firing (8, 9), it is reasonable to propose that pathophysiological low-frequency rhythms, such as those seen in central neuropathic pain, may be mediated by these calcium channels. Nevertheless, this hypothesis has not been directly tested. To determine whether T-type Ca²⁺ channels play a role in the generation of neuropathic pain, thalamocortical oscillatory properties were examined in mice lacking Ca_V3.1 channels following induction of TNP through partial ligation of the inferior orbital nerve (IoN). This channel represents the major T-type Ca²⁺ channel isoform in thalamocortical projection neurons (19). Following IoN ligations, Ca_V3.1 knockout (KO) mice showed significantly attenuated mechanical hypersensitivity, compared with wild-type (WT) mice. Moreover, spectral analysis of thalamocortical rhythms from Ca_V3.1 KO mice showed decreased low-frequency rhythm propensity, compared with WT mice. In addition, response to gamma activation and the spatiotemporal patterns of primary somatosensory (S1) cortex activity were altered in WT but not in KO mice after IoN ligation. Moreover, the cross-frequency interactions between low-frequency and gamma rhythms were significantly increased in WT but not in Ca_V3.1 KO mice. These findings indicate that TNP is associated with altered thalamocortical rhythms, resulting in increased sensitivity to pain as well as pain generation in response to nonnoxious stimuli. In addition, these results indicate that Ca_V3.1 T-type Ca²⁺ channels are fundamentally associated with the alteration of thalamocortical rhythms seen in TNP.     

Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity.Transient receptor potential melastatin 2 (TRPM2) belongs to the TRP protein family and is abundantly expressed in brain neurons, bone marrow, phagocytes, pancreatic β cells, and cardiomyocytes, where it forms Ca²⁺-permeable nonselective cation channels that open under oxidative stress. On contact with pathogens, phagocytic cells produce reactive oxygen species (ROS); the resulting activation of TRPM2 provides the Ca²⁺ influx necessary for cell migration and chemokine production (1). In pancreatic β cells, TRPM2 activity contributes to glucose-evoked insulin secretion; TRPM2 knock-out mice show higher resting blood glucose levels and impaired glucose tolerance (2).TRPM2 activity is also linked to several pathologic conditions that lead to apoptosis (3). Reperfusion after ischemia results in ROS generation; consequent Ca²⁺ influx through TRPM2 causes Ca²⁺ dysregulation and cell death. Certain neurodegenerative diseases, such as Alzheimer’s disease, also involve oxidative stress and TRPM2 activation. In contrast, a loss-of-function TRPM2 mutation identified in patients with amyotrophic lateral sclerosis and Parkinson''s disease dementia (4), as well as two TRPM2 mutations associated with bipolar disorder (5), suggest loss of TRPM2 activity can also cause disease.Similar to most TRP family ion channels, the TRPM2 channel is a homotetramer, and its transmembrane (TM) architecture resembles that of voltage-gated cation channels (6, 7). In addition to the TM domain and an N-terminal cytosolic domain of unknown function, TRPM2 contains an ∼270-residue C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain. The latter shows high (∼50%) sequence homology to the soluble mitochondrial enzyme NUDT9, an active ADP ribose (ADPR) pyrophosphatase (ADPRase) from the Nudix hydrolase family, which splits ADPR into AMP and ribose-5-phosphate (8). TRPM2 channels are coactivated by ADPR binding to NUDT9-H (9) and by Ca²⁺ binding to unidentified intracellular binding sites (10). ADPR is the key that links TRPM2 activation to oxidative stress; in living cells exposed to ROS, ADPR is released from mitochondria (9). In the past, studying TRPM2 channel gating at steady state has been limited by rapid deactivation of TRPM2 currents in cell-free patches (10). This rundown was recently shown to involve a conformational change of the ion selectivity filter, which could be completely prevented by a pore-loop substitution. This “T5L” TRPM2 variant, which shows no rundown but preserves intact regulation of gating by Ca²⁺ and ADPR (11), provides an unprecedented opportunity to study TRPM2 gating at steady state.Early studies reported slow (∼0.1 s⁻¹) but detectable ADPRase activity of isolated purified NUDT9-H (8, 12), classifying TRPM2 into the special group of channel-enzymes (“chanzymes”) that includes TRPM6 and TRPM7 (3) and the CFTR cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel (13). TRPM2 pore opening/closure happens on the timescale of the reported ADPRase activity (11), which is consistent with coupling between gating and catalytic activity, as demonstrated for CFTR in which pore gating follows an irreversible cycle tightly linked to ATP binding and hydrolysis at conserved cytosolic domains (14).The involvement of TRPM2 in multiple diseases has made it an emerging therapeutic target. Depending on the disease, both inhibition (e.g., stroke, myocardial infarction, Alzheimer’s disease, chronic inflammation, hyperinsulinism) and stimulation (e.g., diabetes, amyotrophic lateral sclerosis, Parkinson''s disease dementia, bipolar disorder) of TRPM2 activity might be useful therapeutically. Because TRP family channels are involved in diverse processes (3), any useful TRPM2 agonists/antagonists will need to be highly selective. This singles out the NUDT9-H domain, the component unique to TRPM2, as the most attractive drug target. The significance of understanding whether ADPRase activity and gating are coupled is that optimal strategies for modulating fractional occupancy of a particular conformational state are profoundly different for equilibrium systems than for nonequilibrium systems. For most ion channels, pore gating is an equilibrium process, and open probability is modulated simply by energetic stabilization of either open (activators) or closed (inhibitors) channel ground states. In contrast, channels that gate by a nonequilibrium cycle are most efficiently accumulated in either open or closed states by manipulating the stability of transition states for rate-limiting irreversible steps (15). The aim of this study was to examine the tightness of coupling between the ADPRase cycle and specific gating transitions in TRPM2.     

From the Cover: Purified TMEM16A is sufficient to form Ca2+-activated Cl? channels

Ca²⁺-activated Cl⁻ channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca²⁺ sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca²⁺-dependent Cl⁻ transport with submicromolar sensitivity to Ca²⁺, consistent with what is seen in patch–clamp experiments. The channel is synergistically gated by Ca²⁺ and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca²⁺ dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl⁻ transport with K_is comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.Ca²⁺-activated Cl⁻ channels (CaCCs) regulate a number of physiological functions including epithelial electrolyte secretion in cardiac muscle contractility, preventing polyspermy in oocytes, olfactory responses, and regulating the membrane potential in the nervous system (1, 2). The initial discovery of these channels dates to over 30 y ago in salamander photoreceptors (3) and was followed by numerous reports of Ca²⁺-activated Cl⁻ currents in salivary glands, airway epithelia, Xenopus oocytes, and smooth muscle cells (1). The molecular identity of CaCCs remained unknown until recently, when TMEM16A and -B (also known as Anoctamin1 and 2) were identified as pore-forming components of Ca²⁺-activated Cl⁻ channels (4–6). This seminal discovery allowed researchers to identify the novel roles played by these channels in a variety of physiological processes (2) including mediating salt secretion in kidneys, lungs, and airway epithelia, controlling the excitability of cardiac and smooth muscle cells (7, 8), nociception in sensory neurons (9, 10), and cell proliferation (11–14).Despite these breakthroughs, the TMEM16A channels remain poorly understood at the molecular level, even for their most basic features. Strikingly, two fundamental questions that remain are as follows. (i) Is TMEM16A sufficient to form a functional Ca²⁺-activated channel? Or, is association with ancillary subunits required? (ii) Does Ca²⁺ activate TMEM16A directly or indirectly? Recent work has shown that TMEM16A forms dimers in cells (15–17), associates with members of the ezrin–moesin network of cytoskeletal proteins (18), and that channel activation and ion selectivity require an obligatory association with calmodulin (CaM) (19, 20). It remains unclear, however, whether these proteins are integral components of the functional CaCC complex or whether they are dispensable for function. The basis of the Ca²⁺ sensitivity of TMEM16A is controversial: Several reports suggest that Ca²⁺ modulates TMEM16A indirectly, through association of the channel with calmodulin (19–21). Indeed, the TMEM16A primary sequence lacks recognizable Ca²⁺-binding motifs (22), whereas it possesses at least two putative calmodulin-binding domains (20). In contrast, a conserved pair of acidic residues in the intracellular TM6-7 loop was recently shown to play a key role in the Ca²⁺ sensitivity of TMEM16A and -F (23, 24), suggesting that Ca²⁺ might directly interact with the channel.To address these two questions, we purified the TMEM16A channel and tested its function in vitro. We found that the purified human (h)TMEM16A protein alone recapitulates all of the fundamental biophysical and pharmacological properties of native and heterologously expressed CaCCs. We could not detect a direct interaction between purified TMEM16A and calmodulin. Our results demonstrate that TMEM16A is necessary and sufficient to form functional Ca²⁺-activated Cl⁻ channels and that association with other proteins, such as calmodulin, is not required for their activity.     

Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents

A simultaneous increase in cytosolic Zn²⁺ and Ca²⁺ accompanies the initiation of neuronal cell death signaling cascades. However, the molecular convergence points of cellular processes activated by these cations are poorly understood. Here, we show that Ca²⁺-dependent activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is required for a cell death-enabling process previously shown to also depend on Zn²⁺. We have reported that oxidant-induced intraneuronal Zn²⁺ liberation triggers a syntaxin-dependent incorporation of Kv2.1 voltage-gated potassium channels into the plasma membrane. This channel insertion can be detected as a marked enhancement of delayed rectifier K⁺ currents in voltage clamp measurements observed at least 3 h following a short exposure to an apoptogenic stimulus. This current increase is the process responsible for the cytoplasmic loss of K⁺ that enables protease and nuclease activation during apoptosis. In the present study, we demonstrate that an oxidative stimulus also promotes intracellular Ca²⁺ release and activation of CaMKII, which, in turn, modulates the ability of syntaxin to interact with Kv2.1. Pharmacological or molecular inhibition of CaMKII prevents the K⁺ current enhancement observed following oxidative injury and, importantly, significantly increases neuronal viability. These findings reveal a previously unrecognized cooperative convergence of Ca²⁺- and Zn²⁺-mediated injurious signaling pathways, providing a potentially unique target for therapeutic intervention in neurodegenerative conditions associated with oxidative stress.Calcium has long been recognized as a critical component of neuronal cell death pathways triggered by oxidative, ischemic, and other forms of injury (1). Indeed, Ca²⁺ deregulation has been associated with a variety of detrimental processes in neurons, including mitochondrial dysfunction (2), generation of reactive oxygen species (3), and activation of apoptotic signaling cascades (4). More recently, zinc, a metal crucial for proper cellular functioning (5), has been found to be closely linked to many of the injurious conditions in which Ca²⁺ had been thought to play a prominent role (6–10). In fact, it has been suggested that a number of deleterious properties initially attributed to Ca²⁺ may have significant Zn²⁺-mediated components (11, 12). Although it is virtually impossible to chelate, or remove, Ca²⁺ without disrupting Zn²⁺ levels (13), the introduction of techniques to monitor Ca²⁺ and Zn²⁺ simultaneously in cells (14) has made it increasingly apparent that both cations have important yet possibly distinct roles in neuronal cell death (12, 15–18). However, the relationship between the cell death signaling pathways activated by the cations is unclear, and possible molecular points of convergence between these signaling cascades have yet to be identified.Injurious oxidative and nitrosative stimuli lead to the liberation of intracellular Zn²⁺ from metal binding proteins (19). The released Zn²⁺, in turn, triggers p38 MAPK- and Src-dependent Kv2.1 channel insertion into the plasma membrane, resulting in a prominent increase in delayed rectifier K⁺ currents in dying neurons, with no change in activation voltage, ∼3 h following a brief exposure to the stimulus (20–26). The increase in Kv2.1 channels present in the membrane mediates a pronounced loss of intracellular K⁺, likely accompanied by Cl⁻ (27, 28), that facilitates apoptosome assembly and caspase activation (20, 29–34). Indeed, K⁺ efflux appears to be a requisite event for the completion of many apoptotic programs, including oxidant-induced, Zn²⁺-mediated neuronal death (21).Ca²⁺ has been suggested to regulate the p38 MAPK signaling cascade via Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)-mediated activation of the MAP3K apoptosis signaling kinase-1 (ASK-1) (35). Because ASK-1 is also required for p38-dependent manifestation of the Zn²⁺-triggered, Kv2.1-mediated enhancement of K⁺ currents (36), we hypothesized that the p38 activation cascade may provide a point of convergence between Ca²⁺ and Zn²⁺ signals following oxidative injury. Here, we report that Ca²⁺ and Zn²⁺ signals do, in fact, converge on a cellular event critical for the K⁺ current enhancement, and that CaMKII is required for this process. However, CaMKII does not act upstream of p38 activation as originally hypothesized, but instead interacts with the N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin, which we showed to be necessary for the insertion of Kv2.1-encoded K⁺ channels following an apoptotic stimulus (23).