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The fruit fly Drosophila melanogaster has provided important insights into how sensory information is transduced by transient receptor potential (TRP) channels in the peripheral nervous system (PNS). However, TRP channels alone have not been able to completely model mechanosensitive transduction in mechanoreceptive chordotonal neurons (CNs). Here, we show that, in addition to TRP channels, the sole voltage-gated sodium channel (Na V ) in Drosophila , Para, is localized to the dendrites of CNs. Para is localized to the distal tip of the dendrites in all CNs, from embryos to adults, and is colocalized with the mechanosensitive TRP channels No mechanoreceptor potential C (NompC) and Inactive/Nanchung (Iav/Nan). Para localization also demarcates spike initiation zones (SIZs) in axons and the dendritic localization of Para is indicative of a likely dendritic SIZ in fly CNs. Para is not present in the dendrites of other peripheral sensory neurons. In both multipolar and bipolar neurons in the PNS, Para is present in a proximal region of the axon, comparable to the axonal initial segment (AIS) in vertebrates, 40-60 μm from the soma in multipolar neurons and 20-40 μm in bipolar neurons. Whole-cell reduction of para expression using RNAi in CNs of the adult Johnston's organ (JO) severely affects sound-evoked potentials (SEPs). However, the duality of Para localization in the CN dendrites and axons identifies a need to develop resources to study compartment-specific roles of proteins that will enable us to better understand Para's role in mechanosensitive transduction., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Ravenscroft et al.)

Neuronal atrophy in neurodegenerative diseases is commonly viewed as an early event in a continuum that ultimately results in neuronal loss. In a mouse model of the polyglutamine disorder spinocerebellar ataxia type 1 (SCA1), we tested the hypothesis that cerebellar Purkinje neuron atrophy serves an adaptive role rather than being simply a nonspecific response to injury. In acute cerebellar slices from SCA1 mice, we find that Purkinje neuron pacemaker firing is initially normal but, with the onset of motor dysfunction, becomes disrupted, accompanied by abnormal depolarization. Remarkably, subsequent Purkinje cell atrophy is associated with a restoration of pacemaker firing. The early inability of Purkinje neurons to support repetitive spiking is due to unopposed calcium currents resulting from a reduction in large-conductance calcium-activated potassium (BK) and subthreshold-activated potassium channels. The subsequent restoration of SCA1 Purkinje neuron firing correlates with the recovery of the density of these potassium channels that accompanies cell atrophy. Supporting a critical role for BK channels, viral-mediated increases in BK channel expression in SCA1 Purkinje neurons improves motor dysfunction and partially restores Purkinje neuron morphology. Cerebellar perfusion of flufenamic acid, an agent that restores the depolarized membrane potential of SCA1 Purkinje neurons by activating potassium channels, prevents Purkinje neuron dendritic atrophy. These results suggest that Purkinje neuron dendritic remodeling in ataxia is an adaptive response to increases in intrinsic membrane excitability. Similar adaptive remodeling could apply to other vulnerable neuronal populations in neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Sound encoding is mediated by Ca2+ influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca2+ current through CaV1.3 channels and Ca2+ homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca2+ imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca2+ influx in inner hair cells via fueling Ca2+-ATPases to avoid an increase in cytosolic [Ca2+] and subsequent Ca2+/calmodulin-dependent inactivation of CaV1.3 channels. [ABSTRACT FROM AUTHOR]

The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared with wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2 ) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naive mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Coadministration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. [ABSTRACT FROM AUTHOR]

The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared with wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2 ) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naive mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Coadministration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. [ABSTRACT FROM AUTHOR]

ClC-3 is an intracellular chloride transport protein known to reside on endosomes and synaptic vesicles. The endogenous protein has been notoriously difficult to detect in immunohistological experiments because of the lack of reliable antibodies. Using newly generated antibodies, wenowexamine its expression pattern at the cellular and subcellular level. In all tissues examined, immunostaining indicated that ClC-3 is a vesicular protein, with a prominent expression in endocrine cells like adrenal chromaffin cells and pancreatic islet cells. In line with a possible function of ClC-3 in regulating vesicle trafficking or exocytosis in those secretory cells, capacitance measurements and amperometry indicated that exocytosis of large dense-core vesicles (LDCVs) was decreased in chromaffin cells from ClC-3 knock-out mice. However, immunohistochemistry complemented with subcellular fractionation showed that ClC-3 is not detectable on LDCVs of endocrine cells, but localizes to endosomes and synaptic-like microvesicles in both adrenal chromaffin and pancreatic βcells. This observation points to an indirect influence of ClC-3 on LDCV exocytosis in chromaffin cells, possibly by affecting an intracellular trafficking step. [ABSTRACT FROM AUTHOR]

Ca2+ influx through NMDA receptors (NMDARs) leads to channel inactivation, which limits Ca2+ entry and protects against excitotoxicity. Extensive functional data suggests that this Ca2+-dependent inactivation (CDI) requires both calmodulin (CaM) binding to the C0 cassette of the NR1 subunit's C terminus (CT) and regulation by α-actinin-2, but a molecular understanding of CDI has been elusive. Here we used a number of methods to analyze the molecular nature of the interaction among CaM, α-actinin-2, and the NR1 CT. We found that a single CaM binds to two NR1 CTs in a Ca2+-dependent manner and promotes their reversible "dimerization." Expressed NMDARs containing NR1 concatamers in which the NR1 C termini are "uncoupled" display markedly reduced CDI. In contrast to current models, α-actinin-2 does not bind to the NR1 CT. We propose a new model for CDI in which the noncanonical Ca2+/CaM-dependent dimerization of the two NR1 subunits inactivates the channel by propagating a conformational change from the short NR1 CT to the nearby channel pore. [ABSTRACT FROM AUTHOR]

At most excitatory central synapses, glutamate is released from presynaptic terminals and binds to postsynaptic AMPA receptors, initiating a series of conformational changes that result in ion channel opening. Efficient transmission at these synapses requires that glutamate binding to AMPA receptors results in rapid and near-synchronous opening of postsynaptic receptor channels. In addition, if the information encoded in the frequency of action potential discharge is to be transmitted faithfully, glutamate must dissociate from the receptor quickly, enabling the synapse to discriminate presynaptic action potentials that are spaced closely in time. The current view is that the efficacy of agonists is directly related to the extent to which ligand binding results in closure of the binding domain. For glutamate to dissociate from the receptor, however, the binding domain must open. Previously, we showed that mutations in glutamate receptor subunit 2 that should destabilize the closed conformation not only sped deactivation but also altered the relative efficacy of glutamate and quisqualate. Here we present x-ray crystallographic and single-channel data that support the conclusions that binding domain closing necessarily precedes channel opening and that the kinetics of conformational changes at the level of the binding domain importantly influence ion channel gating. Our findings suggest that the stability of the closed-cleft conformation has been tuned during evolution so that glutamate dissociates from the receptor as rapidly as possible but remains an efficacious agonist. [ABSTRACT FROM AUTHOR]

Submicron local cAMP elevation was used to map the distribution of transduction channels in single olfactory cilia. After the fine fluorescent visualization of the cilium with the laser-scanning confocal microscope, the intraciliary cAMP was jumped locally with the laser beam that photolyzes cytoplasmic caged compounds. Simultaneously, cells' responses were obtained with the whole-cell patch clamp. Responses were observed anywhere within the cilia, showing the broad distribution of transduction channels. For odor detection, such distribution would be useful for expanding the available responding area to increase the quantum efficiency. Also, the stimulus onto only 1 µm region induced > 100 pA response operated by > 700 ~2300 channels, although only 1 pA is sufficient for olfactory cells to generate action potentials. The large local response indicates a presence of strong amplification achieved with a high-density distribution of the transduction channels for the local ciliary excitation. [ABSTRACT FROM AUTHOR]

Serotonin type 3 (5-HT3 ) receptors are members of the pentameric Cys-loop superfamily of receptors that modulate synaptic neurotransmission. In response to agonist binding and unbinding, members of this superfamily undergo a series of conformational transitions that define their functional properties. In this study, we report the results of electrophysiological studies using rapid solution exchange designed to characterize and compare the actions of the high-efficacy agonist serotonin and the low-efficacy agonist dopamine on human 5-HT3A receptors expressed in human embryonic kidney HEK293 cells. In the case of serotonin, receptor activation rates varied with agonist concentration, and deactivation occurred as a single-exponential process with a rate that was similar to the maximal rate of desensitization. Receptors recovered slowly from long desensitizing pulses of serotonin with a sigmoidal time course. In the case of dopamine, receptor activation rates were independent of agonist concentration, receptor deactivation occurred as a complex process that was significantly faster than the maximal rate of desensitization, and recovery from desensitization occurred more quickly than with 5-HT and its time course was not sigmoidal. We developed an allosteric kinetic model for 5-HT3A receptor activation, deactivation, desensitization, and resensitization. Interpretation of our results within the context of this model indicated that the distinct modulatory actions of serotonin versus dopamine are largely attributable to the vastly different rates with which these two agonists induce channel opening and dissociate from open and desensitized states. [ABSTRACT FROM AUTHOR]

In an experimental model of cancer pain, the hyperalgesia that occurs with osteolytic tumor growth is associated with the sensitization of nociceptors. We examined functional and molecular changes in small-diameter dorsal root ganglion (DRG) neurons to determine cellular mechanisms underlying this sensitization. The occurrence of a Ca2+ transient in response to either KCl (25 mM) or capsaicin (500 nM) increased in small neurons isolated from murine L3--L6 DRGs ipsilateral to fibrosarcoma cell tumors. The increased responses were associated with increased mRNA levels for the Ca2+ channel subunit α2δ1 and TRPV1 receptor. Pretreatment with gabapentin, an inhibitor of the β2δ1 subunit, blocked the increased response to KCl in vitro and the mechanical hyperalgesia in tumor-bearing mice in vivo. Similar increases in neuronal responsiveness occurred when DRG neurons from naive mice and fibrosarcoma cells were cocultured for 48 h. The CC chemokine ligand 2 (CCL2) may contribute to the tumor cell-induced sensitization because CCL2 immunoreactivity was present in tumors, high levels of CCL2 peptide were present in microperfusates from tumors, and treatment of DRG neurons in vitro with CCL2 increased the amount of mRNA for the β2δ1 subunit. Together, our data provide strong evidence that the chemical mediator CCL2 is released from tumor cells and evokes phenotypic changes in sensory neurons, including increases in voltage-gated Ca2+ channels that likely underlie the mechanical hyperalgesia in the fibrosarcoma cancer model. More broadly, this study provides a novel in vitro model to resolve the cellular and molecular mechanisms by which tumor cells drive functional changes in nociceptors. [ABSTRACT FROM AUTHOR]

As the Ca2+-sensor for Ca2+-dependent inactivation, calmodulin (CaM) has been proposed, but never definitively demonstrated, to be a constitutive CaV1.2 Ca2+ channel subunit. Here we show that CaM is associated with the CaV1.2 pore-forming α1C subunit in brain in a Ca2+-independent manner. Within its CaM binding pocket, α1C has been proposed to contain a membrane targeting domain. Because ion channel subunits assemble early during channel biosynthesis, we postulated that this association with CaM could afford the opportunity for Ca2+-dependent regulation of membrane targeting. We showed that the isolated domain functioned as a Ca2+/CaM regulated trafficking determinant for CD8 (a model transmembrane protein) using fluorescent-activated cell sorting analysis and, using green fluorescent protein-tagged α1C subunits expressed in cultured hippocampal neurons, that Ca2+/CaM interaction with this domain accelerated trafficking of CaV1.2 channels to distal regions of the dendritic arbor. Furthermore, this Ca2+/CaM-accelerated trafficking was activity dependent. Thus, CaM imparts Ca2+-dependent regulation not only to mature CaV1.2 channels at the cell surface but also to steps during channel biosynthesis. [ABSTRACT FROM AUTHOR]

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (Tb) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)- 6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase Tb of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither Tb nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the Tb response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on Tb. [ABSTRACT FROM AUTHOR]

Pungent chemicals such as allyl isothiocyanate (AITC), cinnamaldehyde, and allicin, produce nociceptive sensation by directly activating transient receptor potential A1 (TRPA1) expressed in sensory afferent neurons. In this study, we found that pungent chemicals added to the pipette or bath solution easily activated TRPA1 in cell-attached patches but failed to do so in inside-out or outside-out patches. Thus, a soluble cytosolic factor was required to activate TRPA1. N-Ethylmaleimide, (2-aminoethyl)-methane thiosulfonate, 2-aminoethoxydiphneyl borate, and trinitrophenol, compounds that are known to activate TRPA1, also failed to activate it in inside-out patches. To identify a factor that supports activation of TRPA1 by pungent chemicals, we screened ∼ 30 intracellular molecules known to modulate ion channels. Among them, pyrophosphate (PPi) and polytriphosphate (PPPi) were found to support activation of TRPA1 by pungent chemicals. Structure--function studies showed that inorganic polyphosphates (polyPn, where n = number of phosphates) with at least four phosphate groups were highly effective (polyP4 ≈ polyP65 ≈ polyP45 ≈ polyP25 > PPPi > PPi), with K½ values ranging from 0.2 to 2.8 mM. Inositol-trisphosphate and inositol-hexaphosphate also partially supported activation of TRPA1 by AITC. ATP, GTP, and phosphatidylinositol-4,5-bisphosphate that have three phosphate groups did not support TRPA1 activation. TRPA1 recorded from cell bodies of trigeminal ganglion neurons showed similar behavior with respect to sensitivity to pungent chemicals; no activation was observed in inside-out patches unless a polyphosphate was present. These results show that TRPA1 requires an intracellular factor to adopt a functional conformation that is sensitive to pungent chemicals and suggest that polyphosphates may partly act as such a factor. [ABSTRACT FROM AUTHOR]

TRPA1 is expressed in primary sensory neurons and hair cells, and it is proposed to be activated by cold stimuli, mechanical stimuli, or pungent ingredients. However, its role in regulating synaptic transmission has never been documented yet. In the present study, we examined whether activation of the TRPA1 channels affects synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. A chief ingredient of mustard oil, allyl isothiocyanate (AITC), superfused for 2 min markedly increased the frequency and amplitude of spontaneous EPSCs (sEPSCs), which was accompanied by an inward current. Similar actions were produced by cinnamaldehyde and allicin. The AITC-induced increases in sEPSC frequency and amplitude were resistant to tetrodotoxin (TTX) and La3+, whereas being significantly reduced in extent in aCa2+-free bath solution. In the presence of glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activities. The AITC-induced increases in sEPSC frequency and amplitude were reduced by ruthenium red, whereas being unaffected by capsazepine. AITC also increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents; this AITC action was abolished in the presence of TTX or glutamate receptor antagonists. These results indicate that TRPA1 appears to be localized not only at presynaptic terminals on SG neurons to enhance glutamate release, but also in terminals of primary afferents innervating onto spinal inhibitory interneurons, which make synapses with SG neurons. This central modulation of sensory signals may be associated with physiological and pathological pain sensations. [ABSTRACT FROM AUTHOR]

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02mMcalcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms. [ABSTRACT FROM AUTHOR]

The accessory α2δ subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel α1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar α2δ-2 subunits with α2δ-2 stably expressed in tsA-201 cells. We identified that α2δ-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel α1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, α2δ-2 is not completely proteolytically processed into α2δ-2 and α-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which α2δ-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/α2δ-2/β4 currents. Furthermore, α2δ-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of α2δ-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable α2δ-2 cell line. In contrast, α2δ-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant α2δ-2 shows reduced functionality in terms of enhancement of CaV2.1/β4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of α2δ-2. Together, these results indicate that both α2δ-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality. [ABSTRACT FROM AUTHOR]

Neurons classified as nociceptors are dependent on nerve growth factor (NGF) during embryonic development, but a large subpopulation lose this dependence during embryonic and postnatal times and become responsive to the transforming growth factor β family member, glial cell line-derived growth factor (GDNF). To elucidate the functional properties of GDNF-dependent nociceptors and distinguish them from nociceptors that retain NGF dependence, the cellular and physiologic properties of sensory neurons of wild-type and transgenic mice that overexpress GDNF in the skin (GDNF-OE) were analyzed using a skin, nerve, dorsal root ganglion, and spinal cord preparation, immunolabeling, and reverse transcriptase-PCR assays. Although an increase in peripheral conduction velocity of C-fibers in GDNF-OE mice was measured, other electrophysiological properties, including resting membrane potential and somal action potentials, were unchanged. We also show that isolectin B4 (IB4)-positive neurons, many of which are responsive to GDNF, exhibited significantly lower thresholds to mechanical stimulation relative to wild-type neurons. However, no change was observed in heat thresholds for the same population of cells. The increase in mechanical sensitivity was found to correlate with significant increases in acid-sensing ion channels 2A and 2B and transient receptor potential channel A1, which are thought to contribute to detection of mechanical stimuli. These data indicate that enhanced expression of GDNF in the skin can change mechanical sensitivity of IB4-positive nociceptive afferents and that this may occur through enhanced expression of specific types of channel proteins. [ABSTRACT FROM AUTHOR]

Transient receptor potential melastatin 8 (TRPM8) and transient receptor potential vanilloid 1 (TRPV1) are ion channels that detect cold and hot sensations, respectively. Their activation depolarizes the peripheral nerve terminals resulting in action potentials that propagate to brain via the spinal cord. These receptors also play a significant role in synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons. Here, we show that TRPM8 is functionally downregulated by activation of protein kinase C (PKC) resulting in inhibition of membrane currents and increases in intracellular Ca2+ compared with upregulation of TRPV1 in cloned and native receptors. Bradykinin significantly downregulates TRPM8 via activation of PKC in DRG neurons. Activation of TRPM8 or TRPV1 at first sensory synapse between DRG and DH neurons leads to a robust increase in frequency of spontaneous/miniature EPSCs. PKC activation blunts TRPM8- and facilitates TRPV1-mediated synaptic transmission. Significantly, downregulation is attributable to PKC-mediated dephosphorylation of TRPM8 that could be reversed by phosphatase inhibitors. These findings suggest that inflammatory thermal hyperalgesia mediated by TRPV1 may be further aggravated by downregulation of TRPM8, because the latter could mediate the much needed cool/soothing sensation. [ABSTRACT FROM AUTHOR]

Ischemic stroke is often accompanied by neuronal hyperexcitability (i.e., seizures), which aggravates brain damage. Therefore, suppressing stroke-induced hyperexcitability and associated excitoxicity is a major focus of treatment for ischemic insults. Both ATP-dependent and Ca2+-activated K+ channels have been implicated in protective mechanisms to suppress ischemia-induced hyperexcitability. Here we provide evidence that the localization and function of Kv2.1, the major somatodendritic delayed rectifier voltage-dependent K+ channel in central neurons, is regulated by hypoxia/ischemia-induced changes in metabolic state and intracellular Ca2+levels. Hypoxia/ ischemia in rat brain induced a dramatic dephosphorylation of Kv2.1 and the translocation of surface Kv2.1 from clusters to a uniform localization. In cultured rat hippocampal neurons, chemical ischemia (CI) elicited a similar dephosphorylation and translocation of Kv2.1. These events were reversible and were mediated by Ca2+ release from intracellular stores and calcineurin-mediated Kv2.1 dephosphorylation. CI also induced a hyperpolarizing shift in the voltage-dependent activation of neuronal delayed rectifier currents (IK), leading to enhanced IK and suppressed neuronal excitability. The IK blocker tetraethylammonium reversed the ischemia-induced suppression of excitability and aggravated ischemic neuronal damage. Our results show that Kv2.1 can act as a novel Ca2+- and metabolic state-sensitive K+ channel and suggest that dynamic modulation of IK/Kv2.1 in response to hypoxia/ischemia suppresses neuronal excitability and could confer neuroprotection in response to brief ischemic insults. [ABSTRACT FROM AUTHOR]

Rad, Gem/Kir, Rem, and Rem2 are members of the Ras-related RGK (Rad, Gem, and Kir) family of small GTP-binding proteins. Heterologous expression of RGK proteins interferes with de novo calcium channel assembly/trafficking and dramatically decreases the amplitude of currents arising from preexisting high-voltage-activated calcium channels. These effects probably result from the direct interaction of RGK proteins with calcium channel β subunits. Among the RGK family, Rem2 is the only member abundantly expressed in neuronal tissues. Here, we examined the ability of Rem2 to modulate endogenous voltage-activated calcium channels in rat sympathetic and dorsal root ganglion neurons. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high-voltage-activated calcium channels without affecting low-voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels required both the Ras homology (core) domain and the polybasic C terminus. Mutation of a putative GTP/Mg2+ binding motif in Rem2 did not affect suppression of calcium currents. Loading neurons with GDP-β-S via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally, [125I]Tyr22-ω-conotoxin GVIA cell surface binding in tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when calcium current was totally abolished. Together, our results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel β subunits in the preassembled N-type channel, thereby forming a nonconducting species. [ABSTRACT FROM AUTHOR]

NMDA receptors (NMDARs) mediate the slow component of excitatory transmission in the CNS and play key roles in synaptic plasticity and excitotoxicity. We investigated the gating reaction mechanism of fully liganded NR1/NR2A recombinant NMDARs (expressed in Xenopus oocytes) by fitting all possible three-closed/two-open-state, noncyclic kinetic schemes to currents elicited by saturating concentrations of glutamate plus glycine. The adequacy of each scheme was assessed by maximum likelihood values and autocorrelation coefficients of single-channel currents, as well as by the predicted time courses of transient macroscopic currents. Two schemes provided the best description for NMDAR gating at both the single-channel and macroscopic levels. These two schemes had coupled open states, only one gateway between the closed and open aggregates, and at least two preopening closed states. These two models could be condensed into a cyclic reaction mechanism. Using a linear reaction scheme, the overall "gating" rates (from the initial stable closed state to the final stable open state) are 177 and 4.4 s-1. [ABSTRACT FROM AUTHOR]

Structurally distinct nicotinic and P2X channels interact functionally, such that coactivation results in cross-inhibition of one or both channel types. It is hypothesized, but not yet proven, that nicotinic and P2X channels interact at the plasma membrane. Here, we show that plasma membrane α4β2 nicotinic and P2X2 channels form a molecular scale partnership and also influence each other when coactivated, resulting in nonadditive cross-inhibitory responses. Total internal reflection fluorescence and fluorescence resonance energy transfer microscopy between fluorescently labeled P2X2 and α4β2 nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X2 and α4β2 channels may form a dimer, with the channels ∼ 80 Å apart. The measurements also show that P2X2 subunits interact specifically and robustly with the β2 subunits in α4β2 channels. The data provide direct evidence for the close spatial apposition of full-length P2X2 and α4β2 channels within 100 nm of the plasma membrane of living cells. [ABSTRACT FROM AUTHOR]

Mechanosensory channels of sensory cells mediate the sensations of hearing, touch, and some forms of pain. The TRPA 1 (a member of the TRP family of ion channel proteins) channel is activated by pain-producing chemicals, and its inhibition impairs hair cell mechanotransduction. As shown here and previously, TRPA1 is expressed by hair cells as well as by most nociceptors (small neurons of dorsal root, trigeminal, and nodose ganglia) and localizes to their sensory terminals (mechanosensory stereocilia and peripheral free nerves, respectively). Thus, TRPA1 channels are proposed to mediate transduction in both hair cells and nociceptors. Accordingly, we find that heterologously expressed TRPA1 display channel behaviors expected for both auditory and nociceptive transducers. First, TRPA1 and the hair cell transducer share a unique set of pore properties not described for any other channel (block by gadolinium, amiloride, gentamicin, and ruthenium red, a ranging conductance of ∼100 pS that is reduced to 54% by calcium, permeating calcium-induced potentiation followed by closure, and reopening by depolarization), supporting a direct role of TRPA1 as a pore-forming subunit of the hair cell transducer. Second, TRPA1 channels inactivate in hyperpolarized cells but remain open in depolarized cells. This property provides a mechanism for the lack of desensitization, coincidence detection, and allodynia that characterize pain by allowing a sensory neuron to respond constantly to sustained stimulation that is suprathreshold (i.e., noxious) and yet permitting the same cell to ignore sustained stimulation that is subthreshold (i.e., innocuous). Our results support a TRPA1 role in both nociceptor and hair cell transduction. [ABSTRACT FROM AUTHOR]

Neurosteroids are produced de novo in neuronal and glial cells, which begin to express steroidogenic enzymes early in development. Studies suggest that neurosteroids may play important roles in neuronal circuit maturation via autocrine and/or paracrine actions. However, the mechanism of action of these agents is not fully understood. We report here that the excitatory neurosteroid pregnenolone sulfate induces a long-lasting strengthening of AMPA receptor-mediated synaptic transmission in rat hippocampal neurons during a restricted developmental period. Using the acute hippocampal slice preparation and patch-clamp electrophysiological techniques, we found that pregnenolone sulfate increases the frequency of AMPA-mediated miniature excitatory postsynaptic currents in CA1 pyramidal neurons. This effect could not be observed in slices from rats older than postnatal day 5. The mechanism of action of pregnenolone sulfate involved a short-term increase in the probability of glutamate release, and this effect is likely mediated by presynaptic NMDA receptors containing the NR2D subunit, which is transiently expressed in the hippocampus. The increase in glutamate release triggered a long-term enhancement of AMPA receptor function that requires activation of postsynaptic NMDA receptors containing NR2B subunits. Importantly, synaptic strengthening could also be triggered by postsynaptic neuron depolarization, and an anti-pregnenolone sulfate antibody scavenger blocked this effect. This finding indicates that a pregnenolone sulfate-like neurosteroid is a previously unrecognized retrograde messenger that is released in an activity-dependent manner during development. [ABSTRACT FROM AUTHOR]

Galantamine (Reminyl; Janssen Pharmaceutica, Titusville, NI) belongs to a class of acetylcholinesterase inhibitors approved for symptomatic treatment of Alzheimer's disease. The drug presumably acts by raising and prolonging the profile of acetylcholine (ACh) via an inhibitory effect on the esterase. However, there is also evidence demonstrating that galantamine can activate the nicotinic ACh receptor or modulate its activation by ACh. In this study, we have examined the ability of galantamine to directly activate the muscle-type nicotinic ACh receptor or to modulate receptor activation by selected nicotinic agonists. Studies of direct activation by galantamine demonstrated that this ligand is a low-efficacy agonist of the muscle-type ACh receptor. Point mutations in the M2-M3 linker (αS269I) and the M2 transmembrane domain (εT264P) had similar effects on receptor activation by galantamine and nicotinic agonists, suggesting that the general features of receptor activation by galantamine are similar to that in the presence of ACh. Experiments performed in the simultaneous presence of galantamine and various nicotinic ligands showed that channel activation by the nicotinic ligands studied (ACh, carbachol, and choline) was not affected by the presence of galantamine at concentrations up to 100 µM. In addition, galantamine did not reduce the initial rate of binding for 125I-α- bungarotoxin. These results demonstrate that galantamine does not interfere with the occupation of the nicotinic agonist binding site by ACh, carbachol, or choline. We conclude that galantamine activates the muscle-type ACh receptor by interacting with a binding site that is distinct from the site for nicotinic agonists. [ABSTRACT FROM AUTHOR]

The subfamily of G-protein-linked inwardly rectifying potassium channels (GIRKs) is coupled to G-protein receptors throughout the CNS and in the heart. We used mutational analysis to address the role of a specific hydrophobic region of the GIRK1 subunit. Deletion of the GIRK1 C-terminal residues 330-384, as well as the point mutation I331R, resulted in a decrease in channel function when coexpressed with GIRK4 in oocytes and in COS-7 cells. Surface protein expression of GIRK1 I331R coexpressed with GIRK4 was comparable with wild type, indicating that subunits assemble and are correctly localized to the membrane. Subsequent mutation of homologous residues in both the GIRK4 subunit and Kir2.1 (Gβy-independent inward rectifier) also resulted in a decrease in channel function. Intracellular domain associations resulted in the coimmunoprecipitation of the GIRK1 N and C termini and GIRK4 N and C termini. The point mutation I331R in the GIRK1 C terminus or L337R in the GIRK4 C terminus decreased the association between the N and C termini. Mutation of a GIRK1 N-terminal hydrophobic residue, predicted structurally to interact with the C-terminal domain, also resulted in a decrease in channel function and termini association. We hypothesize that the hydrophobic nature of this GIRK1 subunit region is critical for interaction between adjacent termini and is permissive for channel gating. In addition, the homologous mutation in cytoplasmic domains of Kir2.1 (L330R) did not disrupt association, suggesting that the overall structural integrity of this region is critical for inward rectifier function. [ABSTRACT FROM AUTHOR]

Acetylcholine binding to muscarinic acetylcholine receptors activates G- proteins, phospholipase C, and protein kinase C (PKC), which phosphorylates brain Na+ channels and reduces peak Na+ current in hippocampal neurons. Because multiple PKC isozymes with different regulatory properties are expressed in hippocampal neurons, we investigated which ones are responsible for mediating this effect. The diacylglycerol analog oleoylacetylglycerol (OAG) reduced the amplitude of Na+ current in dissociated mouse hippocampal neurons by 28.5 ± 5.3% (p < 0.01). The reduction of peak Na+ current was similar with Ca2+-free internal solution and in 92 nM internal Ca2+, suggesting that calcium-dependent, conventional PKC isozymes were unlikely to mediate this response. Gö6976, which inhibits conventional PKC isozymes, reduced the effect of PKC activators only slightly, whereas rottlerin, which inhibits PKCδ preferentially at 5 µM, had no effect. Ro-31- 8425 (20 nM), which inhibits conventional PKC isozymes, did not reduce the response to OAG. However, higher concentrations of Ro-31-8425 (100 nM or 1 µM) that inhibit novel PKC isozymes effectively blocked OAG inhibition of Na+ current. Inclusion of a selective PKC∈-anchoring inhibitor peptide (PKC∈-I) in the recording pipette prevented the reduction of peak Na+ current by OAG, whereas an anchoring inhibitor peptide specific for PKCβ and an inactive scrambled PKC∈-I peptide had no effect. In addition, OAG had no effect on Na+ current in hippocampal neurons from PKC∈ null mice. Overall, our data from four experimental approaches indicate that anchored PKC∈ is the isozyme responsible for PKC-mediated reduction of peak Na+ currents in mouse hippocampal neurons. [ABSTRACT FROM AUTHOR]

ATP-gated P2X2 channels undergo permeability changes through a process that is incompletely understood. In the present study, we used fluorescence resonance energy transfer (FRET) and electrophysiology to measure cytosolic gating motions in P2X2 channels as they enter a state with increased permeability. P2X2 channels underwent permeability changes with a time course that was similar to decreases in FRET between cyan fluorescent protein and yellow fluorescent protein attached to the cytosolic domain of P2X2 channels. Wild-type and mutant channels that did not undergo permeability changes also did not show evidence of cytosolic gating motions. Moreover, immobilizing the cytosolic domain by tethering it to the plasma membrane prevented the switch in permeability and impaired the cytosolic gating motions. Both of these phenotypes were restored when the immobilizing tether was cleaved. The data provide a time-resolved measure of state-specific gating motions and suggest how a cytosolic domain may control ion channel permeability. [ABSTRACT FROM AUTHOR]

A mutation in the sodium channel SCN1A was identified in a small Italian family with dominantly inherited generalized epilepsy with febrile seizures plus (GEFS+). The mutation, D1866Y, alters an evolutionarily conserved aspartate residue in the E-terminal cytoplasmic domain of the sodium channel a subunit. The mutation decreased modulation of the a subunit by β1, which normally causes a negative shift in the voltage dependence of inactivation in oocytes. There was less of a shift with the mutant channel, resulting in a 10 mV difference between the wild-type and mutant channels in the presence of β1. This shift increased the magnitude of the window current, which resulted in more persistent current during a voltage ramp. Computational analysis suggests that neurons expressing the mutant channels will fire an action potential with a shorter onset delay in response to a threshold current injection, and that they will fire multiple action potentials with a shorter interspike interval at a higher input stimulus. These results suggest a causal relationship between a positive shift in the voltage dependence of sodium channel inactivation and spontaneous seizure activity. Direct interaction between the cytoplasmic C-terminal domain of the wild-type a subunit with the β1 or β3 subunit was first demonstrated by yeast two-hybrid analysis. The SCN1A peptide K1846-R1886 is sufficient for β subunit interaction. Coimmunoprecipitation from transfected mammalian cells confirmed the interaction between the C-terminal domains of the a and β1 subunits. The D1866Y mutation weakens this interaction, demonstrating a novel molecular mechanism leading to seizure susceptibility. [ABSTRACT FROM AUTHOR]

Highlights several key advances in the study of the origin and significance of spontaneous firing in the mammalian brain. Biophysical mechanisms underlying spontaneous activity; Plasticity of spontaneous firing in cerebellar Purkinje neurons; Bistability of Purkinje cell output.

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation. [ABSTRACT FROM AUTHOR]

ATP opens ionotropic P2X channels through a process that is poorly understood. We made an array of mutant rat P2X2 channels containing unique alanine substitutions in the transmembrane segments with the goal of identifying possible secondary structure and mapping gating domains in the pore. Alteration of channel function was measured as a change in ATP potency, 2′-3′-O-(4-benzoylbenzoyl)ATP (BzATP) efficacy, and deactivation kinetics. Four mutants (V45A, Y47A, V51A, and D349A) failed to respond to ATP. Seven (H33A, Q37A, I40A, L41A, Y43A, F44A, and I50A) of 22 mutations in the first transmembrane segment (TM1) produced channels with altered potencies, and two mutants (Y43A and F44A) were active in the absence of agonist. The pattern of hits was consistent with a helical secondary structure. In contrast, nine (I328A, P329A, N333A, L338A, T339A, S340A, G342A, G344A, and S345A) of 24 mutations in the second transmembrane segment (TM2) resulted in a change in potency, but no regular pattern of impact was apparent. Many of the same mutations that altered ATP potency also changed the relative efficacy of the partial agonist BzATP. Together, these data suggest that both TM1 and TM2 participate in the conformational changes that occur during receptor activation and help to define domains involved in conformational switching within or near the pore. [ABSTRACT FROM AUTHOR]

Alcohol is an addictive drug that targets a variety of ion channels and receptors. To address whether the effects of alcohol are compartment specific (soma vs dendrite), we examined the effects of ethanol (EtOH) on large-conductance calcium-activated potassium channels (BK) in cell bodies and dendrites of freshly isolated neurons from the rat nucleus accumbens (NAcc), a region known to be critical for the development of addiction. Compartment-specific drug action was indeed observed. Clinically relevant concentrations of EtOH increased somatic but not dendritic BK channel open probability. Electrophysiological single-channel recordings and pharmacological analysis of the BK channel in excised patches from each region indicated a number of differences, suggestive of a compartment-specific expression of the β4 subunit of the BK channel, that might explain the differential alcohol sensitivity. These parameters included activation kinetics, calcium dependency, and toxin blockade. Reverse transcription-PCR showed that both BK channel β1 and β4 subunit mRNAs are found in the NAcc, although the signal for β1 is significantly weaker. Immunohistochemistry revealed that β1 subunits were found in both soma and dendrites, whereas β4 appeared restricted to the soma. These findings suggest that the β4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of β4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated αβ4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter αβ1 BK channel-mediated currents. Finally, an EtOH concentration (50 mM) that increased BK channel open probability strongly decreased the duration of somatic-generated action potential in NAcc neurons. [ABSTRACT FROM AUTHOR]

We used a model system in which dopaminergic (DA) neurons from embryonic rat mesencephalon undergo spontaneous and selective degeneration as they develop in culture. Here, we show that DA cell loss can be prevented efficiently by low concentrations of the Na+ channel agonist veratridine. The survival promoting effect of veratridine was reproduced by, but independent of, glial cell line-derived neurotrophic factor. Neuroprotection by veratridine was exquisitely specific to DA neurons, short-lived after withdrawal, and abolished by tetrodotoxin, indicating that activation of voltage-gated Na+ channels was crucially involved. Calcium measurements showed that veratridine-induced Na+ influx was necessary to maintain intracellular Ca2+ within a neuroprotective range through the stimulation of low-voltage activated T-type calcium channels, a mechanism that was distinct from that elicited by high K+-evoked depolarization. Interestingly, increasing neuronal excitability by treatment with apamin, an inhibitor of Ca2+ - activated K+ channels, or with ouabain, a blocker of the Na+/K+-ATPase pump, was also neuroprotective by a mechanism involving T- type calcium channel activation. These results support the idea that mesencephalic DA neurons depend primarily on excitatory input for their survival during development. [ABSTRACT FROM AUTHOR]

Large-conductance Ca2+ and voltage-regulated K+ channels (Slo1 BK-type) are controlled by two physiological stimuli, membrane voltage and cytosolic Ca2+. Regulation by voltage is similar to that in voltage-dependent K + channels, arising from positively charged amino acids primarily within the $4 transmembrane helices. The basis for regulation by Ca2+ remains controversial. One viewpoint suggests that the extensive cytosolic C terminus contains the Ca2+ regulatory machinery, whereas another suggests that the pore-forming module contains the Ca2+ sensing elements. To address this issue, we take advantage of another Slo family member, the pH-regulated homolog S103. We reason that if the ligand-sensing apparatus is uniquely associated with a particular domain (either the pore or the cytosolic domain), exchange of those domains between Slo1 and Slo3 should result in exchange of ligand dependence in association with the key domain. The results show that the S103 cytosolic module confers pH-dependent regulation on the Slo1 pore module, whereas the Slo1 cytosolic module confers Ca2+-dependent regulation on the S103 pore module. Thus, ligand-specific regulation is defined by interchangeable cytosolic regulatory modules. [ABSTRACT FROM AUTHOR]

Human sensory neuron-specific G-protein-coupled receptors (SNSRs) are expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages. The neuronal function of human SNSRs is difficult to assess because rodent orthologs have yet to be clearly defined, and individual isoforms are found only in a small subset of primary sensory neurons. To circumvent this problem, we expressed human SNSR4 (hSNSR4; also known as Hs.mrgX1) in rat superior cervical ganglion (SCG), dorsal root ganglion (DRG), and hippocampal neurons using nuclear injection or recombinant adenoviruses and examined modulation of ion channels and neurotransmission using whole-cell patch-clamp techniques. BAM8-22 (a 15 amino acid C-terminal fragment of bovine adrenal medulla peptide 22), a peptide agonist derived from proenkephalin, inhibited high (but not low) voltage-activated Ca2+ current in both DRG and SCG neurons expressing hSNSR4, whereas no response was detected in control neurons. The Ca2+ current inhibition was concentration dependent and partially sensitive to Pertussis toxin (PTX) treatment. Additionally, the peptide was highly effective in modulating current arising from M-type K+ channels in SCG neurons expressing hSNSR4. In hippocampal neurons expressing hSNSR4, BAM8-22 induced presynaptic inhibition of transmission that was abolished after PTX treatment. Our data indicate that hSNSR4, when heterologously expressed in rat neurons, can be activated by an opioid-related peptide, couples to Gq/11- proteins as well as PTX-sensitive Gi/o-proteins, and modulates neuronal Ca2+ channels, K+ channels, and synaptic transmission. [ABSTRACT FROM AUTHOR]

Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CAM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Cav1.2 (L-type) Ca22+ channels. CaBP1 interacts directly with the α1 subunit of Cav1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Cav1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Cav1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Cav1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+- dependent inactivation of Cav1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Cav1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons. [ABSTRACT FROM AUTHOR]

Sodium channel β1 subunits modulate α subunit gating and cell surface expression and participate in cell adhesive interactions in vitro. β1 (-/-) mice appear ataxic and display spontaneous generalized seizures. In the optic nerve, the fastest components of the compound action potential are slowed and the number of mature nodes of Ranvier is reduced, but Nav 1.6, contactin, caspr 1, and Kv 1 channels are all localized normally at nodes. At the ultrastructural level, the paranodal septate-like junctions immediately adjacent to the node are missing in a subset of axons, suggesting that β1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Nav 1.1 expression is reduced and Nav1.3 expression is increased in a subset of pyramidal neurons in the CA2/CA3 region, suggesting a basis for the epileptic phenotype. Our results show that β1 subunits play important roles in the regulation of sodium channel density and localization, are involved in axo-glial communication at nodes of Ranvier, and are required for normal action potential conduction and control of excitability in vivo. [ABSTRACT FROM AUTHOR]

Alcohol intoxication alters coordination and motor skills, and this is responsible for a significant number of traffic accident-related deaths around the world. Although the precise mechanism of action of ethanol (EtOH) is presently unknown, studies suggest that it acts, in part, by interfering with normal cerebellar functioning. An important component of cerebellar circuits is the granule cell. The excitability of these abundantly expressed neurons is controlled by the Golgi cell, a subtype of GABAergic interneuron. Granule cells receive GABAergic input in the form of phasic and tonic currents that are mediated by synaptic and extrasynaptic receptors, respectively. Using the acute cerebellar slice preparation and patch-clamp electrophysiological techniques, we found that ethanol induces a parallel increase in both the frequency of spontaneous IPSCs and the magnitude of the tonic current. EtOH (50 mm) did not produce this effect when spontaneous action potentials were blocked with tetrodotoxin. Recordings in the loose-patch cell-attached configuration demonstrated that ethanol increases the frequency of spontaneous action potentials in Golgi cells. Taken together, these findings indicate that ethanol enhances GABAergic inhibition of granule cells via a presynaptic mechanism that involves an increase in action potential-dependent GABA release from Golgi cells. This effect is likely to have an impact on the flow of information through the cerebellar cortex and may contribute to the mechanism by which acute ingestion of alcoholic beverages induces motor impairment. [ABSTRACT FROM AUTHOR]

The slow EPSC (sEPSC) of cerebellar parallel fiber &arr; Purkinje neuron synapses is mediated by metabotropic glutamate receptor type 1 (mGluR1) activation of nonselective cation channels. Here, the channel properties were studied with uniform calibrated photorelease of L-glutamate with ionotropic receptors blocked, allowing isolation of postsynaptic processes, or with parallel fiber stimulation or mGluR 1 agonist application. Evoked current and fluorescence from Ca 2+ indicators were recorded. Noise analysis of the mGluR1 current gave a single-channel conductance of 0.6 pS and showed low open probability at maximal mGluR1 activation. Similar small single-channel conductances were obtained with the mGluR1 agonist (S)-dihydroxyphenylglycine, with parallel fiber or climbing fiber stimulation. The mGluR 1 current fluctuations were unaffected by potassium channel blockers. Photoreleased L-glutamate triggered a Ca 2+ concentration increase in the distal dendrites with a time course similar to that of the mGluR1 current. The proximal dendritic and somatic Ca2+ changes were delayed with respect to the current. Ca2+ channel blockers and the phospholipase Cβ inhibitor 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione, which inhibits mGluR1-activated intracellular Ca 2+ release, did not prevent the dendritic Ca 2+ concentration increase. Polyamine naphthylacetyl spermine and cationic adamantanes that block the pore of the channel were used to vary the mGluR1 current over a wide range in each cell but still at maximal mGluR1 activation. The Ca 2+ influx was inhibited in parallel with the current. The results show that the mGluR1-activated current and the sEPSC are attributable to small-conductance, low-open probability Ca 2+-permeable cation channels that will mediate spine-specific Ca 2+ influx during the parallel fiber sEPSP. [ABSTRACT FROM AUTHOR]

Ca2+ entry through transmitter-gated cation channels, including ATP-gated P2X channels, contributes to an array of physiological processes in excitable and non-excitable cells, but the absolute amount of Ca2+ flowing through P2X channels is unknown. Here we address the issue of precisely how much Ca2+ flows through P2X channels and report the finding that the ATP-gated P2X channel family has remarkably high Ca2+ flux compared with other channels gated by the transmitters ACh, serotonin, protons, and glutamate. Several homomeric and heteromeric P2X channels display fractional Ca2+ currents equivalent to NMDA channels, which hitherto have been thought of as the largest source of transmitter-activated Ca2+ flux. We further suggest that NMDA and P2X channels may use different mechanisms to promote Ca2+ flux across membranes. We find that mutating three critical polar amino acids decreases the Ca2+ flux of P2X2 receptors, suggesting that these residues cluster to form a novel type of Ca2+ selectivity region within the pore. Overall, our data identify P2X channels as a large source of transmitter-activated Ca2+ influx at resting membrane potentials and support the hypothesis that polar amino acids contribute to Ca2+ selection in an ATP-gated ion channel. [ABSTRACT FROM AUTHOR]

Pyramidal neurons in the lateral amygdala discharge trains of action potentials that show marked spike frequency adaptation, which is primarily mediated by activation of a slow calcium-activated potassium current. We show here that these neurons also express an a-dendrotoxin- and tityustoxin-Ka-sensitive voltage-dependent potassium current that plays a key role in the control of spike discharge frequency. This current is selectively targeted to the primary apical dendrite of these neurons. Activation of µ-opioid receptors by application of morphine or D-Ala²-N-Me-Phe4-Glycol5-enkephalin (DAMGO) potentiates spike frequency adaptation by enhancing the α-dendrotoxin-sensitive potassium current. The effects of µ-opioid agonists on spike frequency adaptation were blocked by inhibiting G-proteins with N-ethylmaleimide (NEM) and by blocking phospholipase A2. Application of arachidonic acid mimicked the actions of DAMGO or morphine. These results show that µ-opioid receptor activation enhances spike frequency adaptation in lateral amygdala neurons by modulating a voltage-dependent potassium channel containing Kvl.2 subunits, through activation of the phospholipase A2 arachidonic acid-lipoxygenases cascade. [ABSTRACT FROM AUTHOR]

Circadian oscillators in chicken cone photoreceptors regulate the gating properties of cGMP-gated cationic channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. Here we show that cAMP, acting through protein kinase A (PKA), Ras, and Erk, is part of the circadian output pathway controlling CNGCs. Endogenous and exogenous cAMP cause activation of Erk and Ras, which are more active at night in cones, and increase the apparent affinity of CNGCs for cGMP. The Ras farnesyl transferase inhibitor manumycin-A, and a dominant-negative form of Ras (RasN17) block the circadian rhythms in CNGC gating, as well as the effects of cAMP. A dominant-negative form of the MEK kinase B-Rafalso blocks circadian and cAMP modulation of CNGCs. The circadian output pathway modulating CNGC channels is comprised in part of cAMP → PKA → Ras → B-Raf → MEK → Erk → → CNGCs. cAMP activation of Ras and Erk occur within minutes, whereas modulation of CNGCs requires > 1 hr. However, cAMP protagonists do not alter rhythms in cPer2 mRNA, and their effects on CNGCs cannot be attributed to clock phase-shifting. [ABSTRACT FROM AUTHOR]

The glycine receptor mediates fast synaptic inhibition in the spinal cord and brainstem. Its activation mechanism is not known, despite the physiological importance of this receptor and the fact that it can serve as a prototype for other homopentameric channels. We analyzed single-channel recordings from rat recombinant a 1 glycine receptors by fitting different mechanisms simultaneously to sets of sequences of openings at four glycine concentrations (10-1000 µM). The adequacy of the mechanism and the rate constants thus fitted was judged by examining how well these described the observed dwell-time distributions, open-shut correlation, and single-channel P[subopen] dose-response curve. We found that gating efficacy increased as more glycine molecules bind to the channel, but maximum efficacy was reached when only three (of five) potential binding sites are occupied. Successive binding steps are not identical, implying that binding sites can interact while the channel is shut. These interactions can be interpreted in the light of the topology of the binding sites within a homopentamer. [ABSTRACT FROM AUTHOR]

Neuronal stressors such as hypoxia and firing of action potentials at very high frequencies cause intracellular Na[sup+] to rise and ATP to be consumed faster than it can be regenerated. We report the cloning of a gene encoding a K[sup+] channel, Slick, and demonstrate that functionally it is a hybrid between two classes of K[sup+] channels, Na[sup+]-activated (K[subNa]) and ATP-sensitive (K[subATP]) K[sup+] channels. The Slick channel is activated by intracellular Na[sup+] and Cl[sup-] and is inhibited by intracellular ATP. Slick is widely expressed in the CNS and is detected in heart. We identify a consensus ATP binding site near the C terminus of the channel that is required for ATP and its nonhydrolyzable analogs to reduce open probability. The convergence of Na[sup+], Cl[sup-], and ATP sensitivity in one channel may endow Slick with the ability to integrate multiple indicators of the metabolic state of a cell and to adjust electrical activity appropriately. [ABSTRACT FROM AUTHOR]

Discusses findings about molecules and mechanisms of mechanotransduction. Importance of mechanical energy; Information on transduction channels in hair cells; Proteins involved in mechanotransduction.

Sound encoding is mediated by Ca(2+) influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca(2+) current through CaV1.3 channels and Ca(2+) homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca(2+) imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca(2+) influx in inner hair cells via fueling Ca(2+)-ATPases to avoid an increase in cytosolic [Ca(2+)] and subsequent Ca(2+)/calmodulin-dependent inactivation of CaV1.3 channels., (Copyright © 2014 the authors 0270-6474/14/346843-06$15.00/0.)