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Mechanisms of Fractional Calcium Currents through TRPV1 Channels in Primary Sensory Neurons
Tuesday, February 14, 2017 We inserted BRs originally protected by detergents into liposomes by dialysis and used the charges of liposomes and pH to control the insertion orientation of BR into the liposomes in order to increase the later proton pumping efficiency. We deposited the liposomes with BRs on a porous support to form a supported lipid bilayer (SLB) with BRs. This porous support-SLB-BR structure, which separated platinum cathode from anode, allowed the BRs to pump proton and accumulate electrical potential in a specific direction in response to sunlight. The differences of the proton concentration and electrical potential across the porous support-SLB-BR structure could drive the reduction-oxidation reaction at the electrodes, and therefore generated an electrical current.
Molecular and Cellular Neuroscience 2176-Pos Board B496 Mechanisms of Fractional Calcium Currents through TRPV1 Channels in Primary Sensory Neurons Zhuan Zhou. Institue of Molecular Medicine, Peking University, Beijing, China. TRPV1 and TRPV2 channels are important molecular sensors on the plasma membrane in mammalian physiology (temperature) and diseases (pain) in sensory neurons. These nonselective cation channels permit Ca2þ, Naþ and Kþ influxes simultaneously through its pores to regulate intracellular Ca2þ homeostasis and Ca2þ-dependent neural transmitter release. The Ca2þ influx can be determined by the fractional Ca2þ current (Pf) through a cation channel (Burnashev et al., 1995; Schneggenburger et al., 1993; Yu et al., 2004; Zhou and Neher, 1993). Here, we report (1) Opposite Ca2þ permeability was found as determined by the classic ‘‘Goldman-Hodgkin-Katz equation’’ of PCa/Na (TRPV1 = 7.6 > TRPV2 = 2.8) under non-physiological intra- and extracellular solutions(Caterina et al., 1999; Caterina et al., 1997), or by the ‘‘fractional Ca2þ-current’’ of TRPV1 vs. TRPV2 (Pf = 5.5% vs. 22%) under physiological solutions; (2) The selective filter ‘‘GMGX’’ is similar in TRPV1 and TRPV2, except ‘‘X’’ (X = D for TRPV1 and E for V2). In TRPV1, switching native D to E of TRPV2, Pf(V1, D646E) was greatly increased toward Pf(V2) (from 5.5% to 13%), and vice versa (from 22% to 5.0%); (3) Mutations of two sites outside of the ‘‘GMGX’’, reduced Pf by half; (4) In native neurons replacing TRPV1WT (Pf = 5.5%) with TRPV1-D646E (Pf = 13%), the release mode of Ca2þdependent single vesicle events was dramatically altered from partial (kissand-run) to full release as determined by TIRF-imaging, implicating a physilogical relevance of Pf(TRP) studies. Taken together, TRPV1-D646 (or TRPV2-E604) is the dominant site determining fractional Ca2þ-influx through thermal sensitive TRP channels—a novel mechanism of TRPV channels in presynaptic neural transmitter release for temperature and pain sensation. 2177-Pos Board B497 A Benefit of Randomness in Synaptic Vesicle Release Calvin Zhang1, Charles S. Peskin2. 1 Department of Mathematics, University of Arizona, Tucson, AZ, USA, 2 Courant Institute of Mathematical Sciences, New York University, New York, NY, USA. Noise is not only a source of disturbance, but it also can be beneficial for neuronal information processing. The release of neurotransmitter vesicles in synapses is an unreliable process, especially in the central nervous system. Here we show that the probabilistic nature of neurotransmitter release directly influences the functional role of a synapse, and that a small probability of release per docked vesicle helps reduce the error in the reconstruction of desired signals from the time series of vesicle release events. 2178-Pos Board B498 Glucokinase Mediated Glucosensing in Hypothalamic Neurons Jennifer McFarland, Kendra Seckinger, Mark Rizzo. University of Maryland, Baltimore, Baltimore, MD, USA. Glucosensing is the ability of specialized cells to detect changes in extracellular glucose concentration and convert this information into a change in membrane potential; this allows glucose levels to influence the activity of cells that regulate glucose-altering or glucose-dependent physiological processes. Two major glucosensing cellular populations are pancreatic and hypothalamic cells; the mechanism of glucosensing in pancreatic beta cells is established, but that of hypothalamic neurons remains controversial. Beta cells glucosense by expressing a specialized type of hexokinase (the rate-limiting enzyme in glucose metabolism) called glucokinase (GCK). GCK, unlike standard hexokinase, is not saturated at physiological levels of glucose, allowing glucose metabolism in cells expressing GCK to increase when the extracellular glucose concentration is raised. This increase in metabolism is then converted into a membrane depolarization. Furthermore, S-nitrosylation can enhance the activity of GCK, and therefore enhance glucose metabolism and glucosensing. Here we use GT1-7 cells to explore whether a similar mechanism of glucosensing exists in hypotha-
443a
lamic neurons. The addition of a glucose concentration at which GCK is sensitive (and standard hexokinase is saturated) leads to an increase in cellular metabolism, as measured by NADH autofluorescence; but only in the presence of isoproterenol, a beta-adrenergic receptor agonist which increases intracellular calcium to activate neuronal nitric oxide synthase and facilitate S-nitrosylation. We also use a FRET-based GCK sensor to measure GCK activation under low and high glucose concentrations and in the presence of isoproterenol. These studies reveal the presence of a functional GCK and the sensitivity of GCK to post-translational/signaling cascade modulation. Further, the lack of metabolic response to glucose alone may be linked to high basal activity of GCK in this serum-grown cell line. These results point to a functional, receptor-potentiated glucose sensing mechanism in neurons that is mediated through post-translational activation of GCK. 2179-Pos Board B499 Receptor Level Dissection of Common Versus Discrete Vesicle Release Pathways from Primary Vagal Afferent Terminals James H. Peters. Dept. of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, USA. Primary vagal afferent neurons form strong excitatory glutamatergic synapses onto second-order neurons in the nucleus of the solitary tract and initiate key autonomic and homeostatic reflex pathways. Vesicle fusion and release from vagal central terminals occurs via three distinct pathways including synchronous, asynchronous, and spontaneous. While synchronous and asynchronous release mechanisms require presynaptic action-potential depolarization; spontaneous release is ongoing and independent of action potentials. Evidence suggests these forms of release can be controlled independently indicating discrete and potentially separate vesicle pools and release sites. However, the presence and activation of transient receptor potential (TRP) channels (including TRPV1) at the central terminals dramatically increases the rate of spontaneous vesicle release and eventually diminish action-potential driven synchronous release; consistent with a common releasable pool. Confounding this interpretation is the observation that TRP channel activation also diminishes voltage activated sodium channel signaling. As such the decrease in synchronous release may be a result of action potential failure, rather than depleted vesicle pools. Given that synchronous and spontaneously release glutamate activates both AMPA and NMDA receptors postsynaptically we utilized ligand dependent post-synaptic NMDA receptor blockade (MK-801) to overcome this confound. We found that treatment with MK-801 during exclusively spontaneous release blocked the NMDA component of spontaneous events and subsequent action-potential driven synchronous release. This result demonstrates that glutamate released via spontaneous vesicle release is activating the same population of NMDA receptors as glutamate released synchronously. This finding is consistent with a common release site for synchronous and spontaneous release at vagal afferent terminals. 2180-Pos Board B500 Associative Memory Cells are Recruited to Encode Triple Sensory Signals via Synapse Formation Jin H. Wang, Jing Feng, Wei Lu. Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. Associative learning is a common form of information acquisition, and associative memory is essential for logical reasoning and associative thinking. In addition to memory cells for two associated signals, here we report the recruitment of associative memory cells that encode triple sensory signals. Paired mouse whisker, olfaction and tail stimulations lead to odorant-induced and tailinduced whisker motions. In the mice of showing cross-modal reflexes, barrel cortical neurons and astrocytes become to encode odor and tail signals alongside whisker signal. Such neurons receive the new synapse innervations from the axons of the piriform and S1-tail cortical neurons, in addition to the innate synapse innervations from the axons of the thalamic neurons. The formation of new synapse innervations and the recruitment of associative memory cells require the upregulations of miRNA-324-5p and miRNA-133a-3p that downregulate tau-tubulin kinase-1 (Ttbk1) and methylcytosine dioxygenase Tet3. The associated activations of the sensory cortices elicit their mutual synapse innervations that recruit memory cells to store triple associated signals via epigenetic processes. Furthermore, after the extinction of cross-modal reflexes, the refinements of the synapses and memory cells in the barrel cortex are well maintained, while plasticity in the M1-motor cortex decreases. These results indicate that the acquired signals remain stored in the sensory cortices and the decay of memory retrieval is due to the decreased refinement of their downstream cortical regions in neural circuits from the sensory cortices to the memory-presentation cortices. In terms of the significance of our discoveries, the storages of multiple signals in individual neurons may expand memory
Molecular and Cellular Neuroscience 2176-Pos Board B496 Mechanisms of Fractional Calcium Currents through TRPV1 Channels in Primary Sensory Neurons Zhuan Zhou. Institue of Molecular Medicine, Peking University, Beijing, China. TRPV1 and TRPV2 channels are important molecular sensors on the plasma membrane in mammalian physiology (temperature) and diseases (pain) in sensory neurons. These nonselective cation channels permit Ca2þ, Naþ and Kþ influxes simultaneously through its pores to regulate intracellular Ca2þ homeostasis and Ca2þ-dependent neural transmitter release. The Ca2þ influx can be determined by the fractional Ca2þ current (Pf) through a cation channel (Burnashev et al., 1995; Schneggenburger et al., 1993; Yu et al., 2004; Zhou and Neher, 1993). Here, we report (1) Opposite Ca2þ permeability was found as determined by the classic ‘‘Goldman-Hodgkin-Katz equation’’ of PCa/Na (TRPV1 = 7.6 > TRPV2 = 2.8) under non-physiological intra- and extracellular solutions(Caterina et al., 1999; Caterina et al., 1997), or by the ‘‘fractional Ca2þ-current’’ of TRPV1 vs. TRPV2 (Pf = 5.5% vs. 22%) under physiological solutions; (2) The selective filter ‘‘GMGX’’ is similar in TRPV1 and TRPV2, except ‘‘X’’ (X = D for TRPV1 and E for V2). In TRPV1, switching native D to E of TRPV2, Pf(V1, D646E) was greatly increased toward Pf(V2) (from 5.5% to 13%), and vice versa (from 22% to 5.0%); (3) Mutations of two sites outside of the ‘‘GMGX’’, reduced Pf by half; (4) In native neurons replacing TRPV1WT (Pf = 5.5%) with TRPV1-D646E (Pf = 13%), the release mode of Ca2þdependent single vesicle events was dramatically altered from partial (kissand-run) to full release as determined by TIRF-imaging, implicating a physilogical relevance of Pf(TRP) studies. Taken together, TRPV1-D646 (or TRPV2-E604) is the dominant site determining fractional Ca2þ-influx through thermal sensitive TRP channels—a novel mechanism of TRPV channels in presynaptic neural transmitter release for temperature and pain sensation. 2177-Pos Board B497 A Benefit of Randomness in Synaptic Vesicle Release Calvin Zhang1, Charles S. Peskin2. 1 Department of Mathematics, University of Arizona, Tucson, AZ, USA, 2 Courant Institute of Mathematical Sciences, New York University, New York, NY, USA. Noise is not only a source of disturbance, but it also can be beneficial for neuronal information processing. The release of neurotransmitter vesicles in synapses is an unreliable process, especially in the central nervous system. Here we show that the probabilistic nature of neurotransmitter release directly influences the functional role of a synapse, and that a small probability of release per docked vesicle helps reduce the error in the reconstruction of desired signals from the time series of vesicle release events. 2178-Pos Board B498 Glucokinase Mediated Glucosensing in Hypothalamic Neurons Jennifer McFarland, Kendra Seckinger, Mark Rizzo. University of Maryland, Baltimore, Baltimore, MD, USA. Glucosensing is the ability of specialized cells to detect changes in extracellular glucose concentration and convert this information into a change in membrane potential; this allows glucose levels to influence the activity of cells that regulate glucose-altering or glucose-dependent physiological processes. Two major glucosensing cellular populations are pancreatic and hypothalamic cells; the mechanism of glucosensing in pancreatic beta cells is established, but that of hypothalamic neurons remains controversial. Beta cells glucosense by expressing a specialized type of hexokinase (the rate-limiting enzyme in glucose metabolism) called glucokinase (GCK). GCK, unlike standard hexokinase, is not saturated at physiological levels of glucose, allowing glucose metabolism in cells expressing GCK to increase when the extracellular glucose concentration is raised. This increase in metabolism is then converted into a membrane depolarization. Furthermore, S-nitrosylation can enhance the activity of GCK, and therefore enhance glucose metabolism and glucosensing. Here we use GT1-7 cells to explore whether a similar mechanism of glucosensing exists in hypotha-
443a
lamic neurons. The addition of a glucose concentration at which GCK is sensitive (and standard hexokinase is saturated) leads to an increase in cellular metabolism, as measured by NADH autofluorescence; but only in the presence of isoproterenol, a beta-adrenergic receptor agonist which increases intracellular calcium to activate neuronal nitric oxide synthase and facilitate S-nitrosylation. We also use a FRET-based GCK sensor to measure GCK activation under low and high glucose concentrations and in the presence of isoproterenol. These studies reveal the presence of a functional GCK and the sensitivity of GCK to post-translational/signaling cascade modulation. Further, the lack of metabolic response to glucose alone may be linked to high basal activity of GCK in this serum-grown cell line. These results point to a functional, receptor-potentiated glucose sensing mechanism in neurons that is mediated through post-translational activation of GCK. 2179-Pos Board B499 Receptor Level Dissection of Common Versus Discrete Vesicle Release Pathways from Primary Vagal Afferent Terminals James H. Peters. Dept. of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, USA. Primary vagal afferent neurons form strong excitatory glutamatergic synapses onto second-order neurons in the nucleus of the solitary tract and initiate key autonomic and homeostatic reflex pathways. Vesicle fusion and release from vagal central terminals occurs via three distinct pathways including synchronous, asynchronous, and spontaneous. While synchronous and asynchronous release mechanisms require presynaptic action-potential depolarization; spontaneous release is ongoing and independent of action potentials. Evidence suggests these forms of release can be controlled independently indicating discrete and potentially separate vesicle pools and release sites. However, the presence and activation of transient receptor potential (TRP) channels (including TRPV1) at the central terminals dramatically increases the rate of spontaneous vesicle release and eventually diminish action-potential driven synchronous release; consistent with a common releasable pool. Confounding this interpretation is the observation that TRP channel activation also diminishes voltage activated sodium channel signaling. As such the decrease in synchronous release may be a result of action potential failure, rather than depleted vesicle pools. Given that synchronous and spontaneously release glutamate activates both AMPA and NMDA receptors postsynaptically we utilized ligand dependent post-synaptic NMDA receptor blockade (MK-801) to overcome this confound. We found that treatment with MK-801 during exclusively spontaneous release blocked the NMDA component of spontaneous events and subsequent action-potential driven synchronous release. This result demonstrates that glutamate released via spontaneous vesicle release is activating the same population of NMDA receptors as glutamate released synchronously. This finding is consistent with a common release site for synchronous and spontaneous release at vagal afferent terminals. 2180-Pos Board B500 Associative Memory Cells are Recruited to Encode Triple Sensory Signals via Synapse Formation Jin H. Wang, Jing Feng, Wei Lu. Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. Associative learning is a common form of information acquisition, and associative memory is essential for logical reasoning and associative thinking. In addition to memory cells for two associated signals, here we report the recruitment of associative memory cells that encode triple sensory signals. Paired mouse whisker, olfaction and tail stimulations lead to odorant-induced and tailinduced whisker motions. In the mice of showing cross-modal reflexes, barrel cortical neurons and astrocytes become to encode odor and tail signals alongside whisker signal. Such neurons receive the new synapse innervations from the axons of the piriform and S1-tail cortical neurons, in addition to the innate synapse innervations from the axons of the thalamic neurons. The formation of new synapse innervations and the recruitment of associative memory cells require the upregulations of miRNA-324-5p and miRNA-133a-3p that downregulate tau-tubulin kinase-1 (Ttbk1) and methylcytosine dioxygenase Tet3. The associated activations of the sensory cortices elicit their mutual synapse innervations that recruit memory cells to store triple associated signals via epigenetic processes. Furthermore, after the extinction of cross-modal reflexes, the refinements of the synapses and memory cells in the barrel cortex are well maintained, while plasticity in the M1-motor cortex decreases. These results indicate that the acquired signals remain stored in the sensory cortices and the decay of memory retrieval is due to the decreased refinement of their downstream cortical regions in neural circuits from the sensory cortices to the memory-presentation cortices. In terms of the significance of our discoveries, the storages of multiple signals in individual neurons may expand memory