Renewed scientific interest in sympathetic modulation of muscle and neuromuscular junctions has spurred a flurry of new discoveries with major implications for motor diseases. However, the role sympathetic axons play in the persistent dysfunction that occurs after nerve injuries remains to be explored. Peripheral nerve injuries are common and lead to motor, sensory, and autonomic deficits that result in lifelong disabilities. Given the importance of sympathetic signaling in muscle metabolic health and maintaining bodily homeostasis, it is imperative to understand the regenerative capacity of sympathetic axons after injury. Therefore, we tested sympathetic axon regeneration and functional reinnervation of skin and muscle, both acute and long-term, using a battery of anatomical, pharmacological, chemogenetic, cell culture, analytical chemistry, and electrophysiological techniques. We employed several established growth-enhancing interventions, including electrical stimulation and conditioning lesion, as well as an innovative tool called bioluminescent optogenetics. Our results indicate that sympathetic regeneration is not enhanced by any of these treatments and may even be detrimental to sympathetic regeneration. Despite the complete return of motor reinnervation after sciatic nerve injury, gastrocnemius muscle atrophy and deficits in muscle cellular energy charge, as measured by relative ATP, ADP, and AMP concentrations, persisted long after injury, even with electrical stimulation. We suggest that these long-term deficits in muscle energy charge and atrophy are related to the deficiency in sympathetic axon regeneration. New studies are needed to better understand the mechanisms underlying sympathetic regeneration to develop therapeutics that can enhance the regeneration of all axon types.

N-Methyl-d-aspartate receptors (NMDARs) are a family of ligand-gated ionotropic glutamate receptors that mediate a slow, calcium-permeable component to excitatory neurotransmission. The GluN2D subunit is enriched in GABAergic inhibitory interneurons in cortical tissue. Diminished levels of GABAergic inhibition contribute to multiple neuropsychiatric conditions, suggesting that enhancing inhibition might have therapeutic utility, thus making GluN2D modulation an attractive drug target. Here, we describe the actions of a GluN2C/GluN2D-selective positive allosteric modulator, (+)-EU1180-453, which has improved drug-like properties, such as increased aqueous solubility, in comparison to the first-in-class GluN2C/GluN2D-selective prototypical positive allosteric modulator, (+)-CIQ. (+)-EU1180-453 doubles the NMDAR response at lower concentrations and produces a greater degree of maximal potentiation at 30 µM compared with (+)-CIQ. Using in vitro electrophysiological recordings, we show that (+)-EU1180-453 potentiates triheteromeric NMDARs containing at least one GluN2C or GluN2D subunit and is active at both exon5-lacking and exon5-containing GluN1 splice variants. (+)-EU1180-453 increases glutamate efficacy for GluN2C/GluN2D-containing NMDARs both by prolonging the deactivation time and by potentiating the peak response amplitude. We show that (+)-EU1180-453 selectively increases synaptic NMDAR-mediated charge transfer onto postnatal day 11-15 CA1 stratum radiatum hippocampal interneurons but is without effect on CA1 pyramidal cells. This increased charge transfer enhances inhibitory output from GABAergic interneurons onto CA1 pyramidal cells in a GluN2D-dependent manner. (+)-EU1180-453 also shifts excitatory-to-inhibitory coupling towards increased inhibition and produces enhanced gamma-band power from carbachol-induced field potential oscillations in hippocampal slices. Thus, (+)-EU1180-453 can enhance overall circuit inhibition, which could prove therapeutically useful for the treatment of anxiety, depression, schizophrenia and other neuropsychiatric disorders. KEY POINTS: (+)EU-1180-453 is a GluN2C/GluN2D positive allosteric modulator and is active at triheteromeric receptors. (+)EU-1180-453 is active at exon5-containing and exon5-lacking GluN1-containing receptors. (+)EU-1180-453 selectively potentiates the interneuron network and can enhance carbachol-induced gamma-band power.

N-Methyl-d-aspartate receptors (NMDARs) are known for their role in mediating a calcium-permeable, slow component of excitatory synaptic transmission. These receptors play important roles in multiple facets of brain functions, and their dysfunction has been implicated in neurological disease aetiology. Here, we describe the actions of a positive allosteric modulator (PAM), EU1622-240, on NMDARs within the hippocampal circuit. EU1622-240 is a pan-PAM that enhances the function of all GluN2 subunit-containing NMDARs with submicromolar potency, with the strongest effects on GluN2C- and GluN2D-containing NMDARs. Previously, we have shown that EU1622-240 enhances the maximal response, prolongs the response time course, enhances agonist potency and reduces single channel conductance. Using whole-cell patch-clamp recordings, we evaluated the effects of this PAM on both CA1 pyramidal cells and CA1 stratum radiatum interneurons in immature hippocampus. Although we observed potentiation of evoked NMDAR-mediated EPSCs on both CA1 pyramidal cells and interneurons, the PAM preferentially enhanced interneuron excitability owing to the expression of GluN2D in interneurons and increased the ratio of inhibition to excitation. This appears to result from cellular depolarization, increased spike firing and enhanced NMDAR-mediated current charge transfer in interneurons. In contrast, EU1622-240 did not detectably depolarize CA1 pyramidal cells in slices but did have modest effects when bicuculline was used to block GABAergic signalling. We also observed EU1622-240 enhancement of AMPA receptor synaptic signalling in a manner reminiscent of long-term potentiation. These data support the idea that EU1622-240 enhances interneuron function, with modest effects on the CA1 pyramidal cells, providing therapeutically beneficial effects in situations where interneuron output is diminished. KEY POINTS: EU1622-240 is a potent positive allosteric modulator of all GluN2-containing NMDA receptors. EU1622-240 is active at native receptors in acute brain slices, increasing NMDA receptor-mediated charge transfer onto both CA1 principal cells and interneurons. Despite its actions on principal cells, EU1622-240 appears to drive preferential enhancement of interneuron function within the hippocampal network. EU1622-240 is also capable of increasing calcium flow into cultured hippocampal neurons, in addition to influencing AMPA receptor-mediated EPSPs that occlude conventional theta-burst-driven long-term potentiation.

The TMEM16A calcium-activated chloride channel is a promising therapeutic target for various diseases. Niclosamide, an anthelmintic medication, has been considered as a TMEM16A inhibitor for treating asthma and chronic obstructive pulmonary disease, but was recently found to possess broad-spectrum off-target effects. Here we show that, under physiological conditions, niclosamide acutely potentiates TMEM16A without having any inhibitory effect. Our computational and functional characterizations pinpoint a putative niclosamide binding site on the extracellular side of TMEM16A. Mutations in this site attenuate the potentiation. Moreover, niclosamide potentiates endogenous TMEM16A in vascular smooth muscle cells, triggers intracellular calcium increase, and constricts the murine mesenteric artery. Our findings advise caution when considering niclosamide as a TMEM16A inhibitor to treat diseases such as asthma, COPD, and hypertension. The identification of the putative niclosamide binding site provides insights into the mechanism of TMEM16A pharmacological modulation, shining light on developing specific TMEM16A modulators to treat human diseases.

Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology(1-4). Here we uncover a directional permeability of BEST2 to glutamate that heavily favours glutamate exit, identify glutamine synthetase (GS) as a binding partner of BEST2 in the ciliary body of the eye, and solve the structure of the BEST2-GS complex. BEST2 reduces cytosolic GS activity by tethering GS to the cell membrane. GS extends the ion conducting pathway of BEST2 through its central cavity and inhibits BEST2 channel function in the absence of intracellular glutamate, but sensitizes BEST2 to intracellular glutamate, which promotes the opening of BEST2 and thus relieves the inhibitory effect of GS. We demonstrate the physiological role of BEST2 in conducting chloride and glutamate and the influence of GS in non-pigmented ciliary epithelial cells. Together, our results reveal a novel mechanism of glutamate release through BEST2-GS.

TMEM16A is a Ca2+-activated Cl− channel that regulates diverse cellular functions including fluid secretion, neuronal excitability and smooth muscle contraction. TMEM16A is activated by cytosolic Ca2+ and modulated by binding of the signaling lipid, PIP2. However, it remains elusive how PIP2 binds the channel and how the binding affects molecular events underlying transport in the protein. Here, we use extended molecular dynamics (MD) simulations coupled with electrophysiology, mutagenesis, and functional assays, to characterize PIP2 binding modes and sites in TMEM16A and the conformational response of the channel to PIP2 binding. To enhance sampling of lipid-protein interactions, multiple independent simulations were performed using the highly mobile membrane mimetic (HMMM) lipid bilayers (including 1.4% PIP2). Owing to the rapid lipid rearrangement in HMMM, spontaneous bindings of PIP2 to eight potential sites on the channel were captured. Three of the sites account for 85% of the observed PIP2-TMEM16A interactions and, importantly, were validated to be critical for PIP2 regulation through mutagenesis experiments. To determine protein dynamics in response to PIP2 binding, additional equilibrium simulations were performed after conversion of the HMMM models to full membranes. Intriguingly, PIP2 binding alters the conformation of the cytoplasmic extension of transmembrane helix 6 (TM6), which forms one side of the channel pore and plays a crucial role in channel gating. The pore dilation induced by the rotation of TM6 increases the accessibility of the inner vestibule to cytosolic ions and results in spontaneous penetration of Cl- ions into the pore. These data add to a growing body of knowledge showing that TMEM16A is a highly allosteric protein gated by a network of interactions involving PIP2 lipids.

Amazing Anoctamins All Around. Bamboozling Biologists. Carrying Chloride Currents. Depolarizing DRGs. Enabling Epithelial Effluxes. Flip-Flopping Fat. Generating Gut Gurgling. Honing Hypertension. Importing Iodide. Juxtaposing Junctions. Knotting Kidneys. Liquidating Lachryma. Mending Muscle Membranes. Nourishing Negativity. Ombambulating Olfaction. Palming PIP2. Quenching Q10. Reducing Resistance. Scrambling Supermolecules. Tagging Tumors. Ushering UPS. Vasoconstricting Vessels. Wetting Windpipes. X-ing Xenopus. Yuppifying Yeast. In my talk, I will begin by explaining some of these functions and activities of Anoctamins (also known as TMEM16s) and then will focus on recent work from my lab on regulation of ANO1 by phosphatidylinositol (4,5) bisphosphate and the role of ANO5 in membrane repair. And, I will offer a prize for the first person to offer a good phrase starting with the letter “Z”.

Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is a low-abundance phospholipid in inner leaflet of the plasma membrane that has a remarkable number of functions in cell physiology including cytoskeletal organization, membrane trafficking, and ion channel gating. While the regulation of cation channels by phosphoinositides has been studied extensively, relatively little is known about the effects of phosphoinositides on anion channel function. We have previously used a combination of patch-clamp electrophysiology and atomistic molecular-dynamics (MD) simulations to identify PI(4,5)P2 binding modes and sites on ANO1. ANO1 (TMEM16A) is a Ca2+-activated chloride channel that controls a wide range of functions encompassing electrical excitability, blood pressure, gut motility, fluid and electrolyte secretion, and other functions. PI(4,5)P2 has at least two different effects on ANO1 currents. ANO1 currents activated by sub-micromolar Ca2+ concentrations in excised inside-out patches are increased in amplitude by the addition of PI(4,5)P2 in a voltage-dependent manner with an EC50=1.24 µM at 100 mV. In contrast, saturating Ca2+ concentrations first activate ANO1 currents, but the currents then rapidly inactivate with time. This Ca2+-dependent inactivation is slowed significantly when PI(4,5)P2 is added to the excised patch. Mutagenesis of basic amino acids near the membrane-cytosol interface identified 3 regions of the protein that are critical for PI(4,5)P2 regulation. These sites correspond to sites identified by unbiased atomistic molecular dynamics simulations. These binding sites form a band at the cytosolic interface of the membrane. Here, we explore the effects of PI(4,5)P2 binding to its different binding sites on ANO1 function. Specifically, we examine how PI(4,5)P2 binding to different sites on ANO1 affect the stimulation of current amplitude at low Ca2+ concentration and current inactivation at high Ca2+ concentration. Our findings provide insights into the mechanism understanding the complicated physiological phenomenon of ANO1 channel gating.

ANO1 (TMEM16A) is a Ca2+-activated Cl− channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Here we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2. Three of these sites captured 85% of all ANO1 - PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found three regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same three sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid sidechains and phosphate/hydroxyl groups on PI(4,5)P2. Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl−ions. We propose a model consisting of a network of three PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating. Significance statement Membrane proteins dwell in a sea of phospholipids that not only structurally stabilize the proteins by providing a hydrophobic environment for their transmembrane segments, but also dynamically regulate protein function. While many cation channels are known to be regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), relatively little is known about anion channel regulation by phosphoinositides. Using a combination of patch clamp electrophysiology and atomistic molecular dynamics simulations, we have identified several PI(4,5)P2 binding sites in ANO1 (TMEM16A), a Cl− channel that performs myriad physiological functions from epithelial fluid secretion to regulation of electrical excitability. These binding sites form a band at the cytosolic interface of the membrane that we propose constitute a network to dynamically regulate this highly allosteric protein.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.

Neurons of the CNS elaborate highly branched dendritic arbors that host numerous dendritic spines, which serve as the postsynaptic platform for most excitatory synapses. The actin cytoskeleton plays an important role in dendrite development and spine formation, but the underlying mechanisms remain incompletely understood. Tropomodulins (Tmods) are a family of actin-binding proteins that cap the slow-growing (pointed) end of actin filaments, thereby regulating the stability, length, and architecture of complex actin networks in diverse cell types. Three members of the Tmod family, Tmod1, Tmod2, and Tmod3 are expressed in the vertebrate CNS, but their function in neuronal development is largely unknown. In this study, we present evidence that Tmod1 and Tmod2 exhibit distinct roles in regulating spine development and dendritic arborization, respectively. Using rat hippocampal tissues from both sexes, we find that Tmod1 and Tmod2 are expressed with distinct developmental profiles: Tmod2 is expressed early during hippocampal development, whereas Tmod1 expression coincides with synaptogenesis. We then show that knockdown of Tmod2, but not Tmod1, severely impairs dendritic branching. Both Tmod1 and Tmod2 are localized to a distinct subspine region where they regulate local F-actin stability. However, the knockdown of Tmod1, but not Tmod2, disrupts spine morphogenesis and impairs synapse formation. Collectively, these findings demonstrate that regulation of the actin cytoskeleton by different members of the Tmod family plays an important role in distinct aspects of dendrite and spine development. SIGNIFICANCE STATEMENT The Tropomodulin family of molecules is best known for controlling the length and stability of actin myofilaments in skeletal muscles. While several Tropomodulin members are expressed in the brain, fundamental knowledge about their role in neuronal function is limited. In this study, we show the unique expression profile and subcellular distribution of Tmod1 and Tmod2 in hippocampal neurons. While both Tmod1 and Tmod2 regulate F-actin stability, we find that they exhibit isoform-specific roles in dendrite development and synapse formation: Tmod2 regulates dendritic arborization, whereas Tmod1 is required for spine development and synapse formation. These findings provide novel insight into the actin regulatory mechanisms underlying neuronal development, thereby shedding light on potential pathways disrupted in a number of neurological disorders.

Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell-cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca2+-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5(-/-) mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca2+-dependent phosphatidylserine exposure on the surface of Ano5(-/-) MPCs and a decrease in the amplitude of Ca2+-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5(-/-) MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca2+-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.

From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters. Disruption of this asymmetry by ATP-independent phospholipid scrambling is important in cellular signaling, but its mechanism remains incompletely understood. Using MD simulations coupled with experimental assays, we show that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer on the fungal scramblase nhTMEM16 serves as the pathway for both lipid translocation and ion conduction across the membrane. Ca2+ binding stimulates its open conformation by altering the structure of transmembrane helices that line the cavity. We have identified key amino acids necessary for phospholipid scrambling and validated the idea that ions permeate TMEM16 Cl- channels via a structurally homologous pathway by showing that mutation of two residues in the pore region of the TMEM16A Ca2+-activated Cl- channel convert it into a robust scramblase.

Phospholipid scrambling (PLS) is a ubiquitous cellular mechanism involving the regulated bidirectional transport of phospholipids down their concentration gradient between membrane leaflets. ANO6/TMEM16F has been shown to be essential for Ca(2+)-dependent PLS, but controversy surrounds whether ANO6 is a phospholipid scramblase or an ion channel like other ANO/TMEM16 family members. Combining patch clamp recording with measurement of PLS, we show that ANO6 elicits robust Ca(2+)-dependent PLS coinciding with ionic currents that are explained by ionic leak during phospholipid translocation. By analyzing ANO1-ANO6 chimeric proteins, we identify a domain in ANO6 necessary for PLS and sufficient to confer this function on ANO1, which normally does not scramble. Homology modeling shows that the scramblase domain forms an unusual hydrophilic cleft that faces the lipid bilayer and may function to facilitate translocation of phospholipid between membrane leaflets. These findings provide a mechanistic framework for understanding PLS and how ANO6 functions in this process.

Anoctamin1 (ANO1) encodes a Ca2+‐activated chloride (Cl−) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1‐encoded CaCCs in ischemia‐induced arrhythmias in the heart. Quantitative real‐time RT‐PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch‐clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca2+‐activated Cl− current (ICl.Ca) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of ICl.Ca, which was inhibited by a specific ANO1 inhibitor, T16Ainh‐A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia‐induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper “spike and dome” compared to control cardiomyocytes from non‐ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia‐induced early phase 1 repolarization acceleration and caused a much shallower “spike and dome”. We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia‐induced increase in ANO1 expression may be responsible for the increased density of ICl.Ca in the ischemic heart and may contribute, at least in part, to ischemia‐induced arrhythmias. J. Cell. Physiol. 230: 337–346, 2015. © 2014 Wiley Periodicals, Inc.