A Novel Ca2+ Influx Pathway in Mammalian Primary Sensory Neurons Is Activated by Caffeine

2001 ◽  
Vol 86 (1) ◽  
pp. 190-196 ◽  
Author(s):  
Robert E. Hoesch ◽  
Daniel Weinreich ◽  
Joseph P. Y. Kao
Keyword(s):  
Sensory Neurons ◽  
Ryanodine Receptors ◽  
Reversal Potential ◽  
Significant Rise ◽  
Adult Rabbit ◽  
Pharmacological Agents ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Intracellular Ca ◽  
Ganglion Neurons

Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca2+ influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca2+-induced Ca2+ release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca2+ concentration ([Ca2+]i) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca2+stores or pharmacological blockade of intracellular Ca2+ release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca2+]i in ∼50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca2+ was obligatory for such caffeine-induced [Ca2+]i rises in this population of NGNs, suggesting that Ca2+ influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca2+]i. The inward current had a reversal potential of +8.1 ± 6.1 (SE) mV ( n = 4), a mean peak amplitude of −126 ± 24 pA ( n = 4) at E m = −50 mV, and a slope conductance of 1.43 ± 0.79 nS ( n= 4). Estimated EC50 values for caffeine-induced CICR and for caffeine-activated current were 1.5 and ∼0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca2+]i, in the presence of extracellular Ca2+, can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca2+ influx pathway.

Localized IP3-Evoked Ca2+ Release Activates a K+ Current in Primary Vagal Sensory Neurons

2004 ◽  
Vol 91 (5) ◽  
pp. 2344-2352 ◽  
Author(s):  
Robert E. Hoesch ◽  
Daniel Weinreich ◽  
Joseph P. Y. Kao
Keyword(s):  
Sensory Neurons ◽  
Reversal Potential ◽  
Nodose Ganglion ◽  
Ion Concentration ◽  
Equilibrium Potential ◽  
Nodose Ganglion Neurons ◽  
Patch Pipette ◽  
Intracellular Ca ◽  
K Current ◽  
Ganglion Neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP3, and the consequent release of Ca2+, could trigger a Ca2+-activated K+ current ( IIP3). Photorelease of caged IP3 evoked an IIP3 that averaged 2.36 ± 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at –50 mV. IIP3 was abolished by intracellular BAPTA (2 mM), a Ca2+ chelator. Changing the K+ equilibrium potential by increasing extracellular K+ ion concentration caused a predicted Nernstian shift in the reversal potential of IIP3. These results indicated that IIP3 was a Ca2+-dependent K+ current. IIP3 was unaffected by three common antagonists of Ca2+-activated K+ currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 μM) included in the patch pipette. We have previously demonstrated that both IP3-evoked Ca2+ release and Ca2+-induced Ca2+ release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca2+-activated K+ current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca2+ stores, we showed that IP3-evoked Ca2+ release occurs independently of CICR, but interestingly, that a component of IIP3 requires CICR. These data suggest that IP3-evoked Ca2+ release activates a K+ current that is pharmacologically distinct from other Ca2+-activated K+ currents in NGNs. We describe several models that explain our results based on Ca2+ signaling microdomains in NGNs.

scholarly journals Voltage-gated Na+ currents in human dorsal root ganglion neurons

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Xiulin Zhang ◽  
Birgit T Priest ◽  
Inna Belfer ◽  
Michael S Gold
Keyword(s):  
Sensory Neurons ◽  
Tissue Injury ◽  
Dorsal Root Ganglion Neurons ◽  
Pharmacological Properties ◽  
Pain Mechanisms ◽  
Voltage Gated ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Ganglion Neurons ◽  
Peripheral Sensory

Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms.

Multiple Effects of Serotonin and Acetylcholine on Hyperpolarization-Activated Inward Current in Locomotor Activity-Related Neurons in Cfos-EGFP Mice

2010 ◽  
Vol 104 (1) ◽  
pp. 366-381 ◽  
Author(s):  
Yue Dai ◽  
Larry M. Jordan
Keyword(s):  
Spinal Cord ◽  
Motor System ◽  
Reversal Potential ◽  
Rhythmic Activity ◽  
Maximal Conductance ◽  
Inward Current ◽  
Spinal Interneurons ◽  
Whole Cell Patch Clamp ◽  
Activation Rate ◽  
Maximal Activation

Hyperpolarization-activated inward current ( Ih) has been shown to be involved in production of bursting during various forms of rhythmic activity. However, details of Ih in spinal interneurons related to locomotion remain unknown. Using Cfos-EGFP transgenic mice (P6–P12) we are able to target the spinal interneurons activated by locomotion. Following a locomotor task, whole cell patch-clamp recordings were obtained from ventral EGFP+ neurons in spinal cord slices (T13–L4, 200–250 μm). Ih was found in 51% of EGFP+ neurons ( n = 149) with almost even distribution in lamina VII (51%), VIII (47%), and X (55%). Ih could be blocked by ZD7288 (10–20 μM) or cesium (1–1.5 mM) but was insensitive to barium (2–2.5 mM). Ih activated at −80.1 ± 9.2 mV with half-maximal activation −95.5 ± 13.3 mV, activation rate 10.0 ± 3.2 mV, time constant 745 ± 501 ms, maximal conductance 1.0 ± 0.7 nS, and reversal potential −34.3 ± 3.6 mV. 5-HT (15–20 μM) and ACh (20–30 μM) produced variable effects on Ih. 5-HT increased Ih in 43% of EGFP+ neurons ( n = 37), decreased Ih in 24%, and had no effect on Ih in 33% of the neurons. ACh decreased Ih in 67% of EGFP+ neurons ( n = 18) with unchanged Ih in 33% of the neurons. This study characterizes the Ih in locomotor-related interneurons and is the first to demonstrate the variable effects of 5-HT and ACh on Ih in rodent spinal interneurons. The finding of 5-HT and ACh-induced reduction of Ih in EGFP+ neurons suggests a novel mechanism that the motor system could use to limit the participation of certain neurons in locomotion.

Mitochondrial Ca2+ Activates a Cation Current in Aplysia Bag Cell Neurons

2010 ◽  
Vol 103 (3) ◽  
pp. 1543-1556 ◽  
Author(s):  
Charlene M. Hickey ◽  
Julia E. Geiger ◽  
Chris J. Groten ◽  
Neil S. Magoski
Keyword(s):  
Endoplasmic Reticulum ◽  
Ion Channels ◽  
Time Course ◽  
Reversal Potential ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Inward Current ◽  
Cation Current ◽  
Intracellular Ca ◽  
Bag Cell Neurons

Ion channels may be gated by Ca2+ entering from the extracellular space or released from intracellular stores—typically the endoplasmic reticulum. The present study examines how Ca2+ impacts ion channels in the bag cell neurons of Aplysia californica. These neuroendocrine cells trigger ovulation through an afterdischarge involving Ca2+ influx from Ca2+ channels and Ca2+ release from both the mitochondria and endoplasmic reticulum. Liberating mitochondrial Ca2+ with the protonophore, carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone (FCCP), depolarized bag cell neurons, whereas depleting endoplasmic reticulum Ca2+ with the Ca2+-ATPase inhibitor, cyclopiazonic acid, did not. In a concentration-dependent manner, FCCP elicited an inward current associated with an increase in conductance and a linear current/voltage relationship that reversed near −40 mV. The reversal potential was unaffected by changing intracellular Cl−, but left-shifted when extracellular Ca2+ was removed and right-shifted when intracellular K+ was decreased. Strong buffering of intracellular Ca2+ decreased the current, although the response was not altered by blocking Ca2+-dependent proteases. Furthermore, fura imaging demonstrated that FCCP elevated intracellular Ca2+ with a time course similar to the current itself. Inhibiting either the V-type H+-ATPase or the ATP synthetase failed to produce a current, ruling out acidic Ca2+ stores or disruption of ATP production as mechanisms for the FCCP response. Similarly, any involvement of reactive oxygen species potentially produced by mitochondrial depolarization was mitigated by the fact that dialysis with xanthine/xanthine oxidase did not evoke an inward current. However, both the FCCP-induced current and Ca2+ elevation were diminished by disabling the mitochondrial permeability transition pore with the alkylating agent, N-ethylmaleimide. The data suggest that mitochondrial Ca2+ gates a voltage-independent, nonselective cation current with the potential to drive the afterdischarge and contribute to reproduction. Employing Ca2+ from mitochondria, rather than the more common endoplasmic reticulum, represents a diversification of the mechanisms that influence neuronal activity.

scholarly journals L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons

2016 ◽  
Vol 310 (11) ◽  
pp. R1120-R1127 ◽  
Author(s):  
Miloslav Kolaj ◽  
Li Zhang ◽  
Leo P. Renaud
Keyword(s):  
Transient Receptor Potential ◽  
Cannabinoid Receptor ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
Receptor Potential ◽  
Thyrotropin Releasing Hormone ◽  
Induced Response ◽  
Inward Current ◽  
Releasing Hormone ◽  
Intracellular Ca

In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca2+) contribution to the components of this TRH-induced response. TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca2+]o, suggesting a critical role for both intracellular Ca2+ release and Ca2+ influx. TRH-induced inward current was unchanged by T-type Ca2+ channel blockade, but was decreased by blockade of high-voltage-activated Ca2+ channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca2+ influx via L-type Ca2+ channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca2+-induced Ca2+ release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca2+ influx via opening of L-type Ca2+ channels, a sensitivity of a TRPC-like component to intracellular Ca2+ release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component.

Mitochondrial Modulation of Ca2+-Induced Ca2+-Release in Rat Sensory Neurons

2006 ◽  
Vol 96 (3) ◽  
pp. 1093-1104 ◽  
Author(s):  
Joshua G. Jackson ◽  
Stanley A. Thayer
Keyword(s):  
Plasma Membrane ◽  
Sensory Neurons ◽  
Oscillation Frequency ◽  
Cyclopiazonic Acid ◽  
Cellular Level ◽  
Dorsal Root Ganglion Neurons ◽  
Whole Cell Patch Clamp ◽  
Carbonyl Cyanide ◽  
Synthase Inhibitor ◽  
Ganglion Neurons

Ca2+-induced Ca2+-release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.

TMEM16F is a component of a Ca2+-activated Cl− channel but not a volume-sensitive outwardly rectifying Cl− channel

2013 ◽  
Vol 304 (8) ◽  
pp. C748-C759 ◽  
Author(s):  
Takahiro Shimizu ◽  
Takahiro Iehara ◽  
Kaori Sato ◽  
Takuto Fujii ◽  
Hideki Sakai ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Reversal Potential ◽  
Bathing Solution ◽  
Osmotic Swelling ◽  
Cl Channel ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Intracellular Ca ◽  
Current Voltage Relationship ◽  
Voltage Relationship

TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl− channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca2+ ionophore, ionomycin, or by an increase in the intracellular free Ca2+ concentration. The free Ca2+ concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca2+ level, in contrast to TMEM16A/B currents. The Ca2+-activated TMEM16F currents were anion selective, because replacing Cl− with aspartate− in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I− > Br− > Cl− > F− > aspartate−. Niflumic acid, a Ca2+-activated Cl− channel blocker, inhibited the TMEM16F-dependent Cl− currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl− channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca2+-activated Cl− channel with a Ca2+ sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.

Modulation of Voltage-Activated Calcium Currents by Mechanical Stimulation in Rat Sensory Neurons

1998 ◽  
Vol 80 (4) ◽  
pp. 1647-1652 ◽  
Author(s):  
Yona Bouskila ◽  
Hugh Bostock
Keyword(s):  
Action Potential ◽  
Sensory Neurons ◽  
Mechanical Stimulation ◽  
Action Potential Duration ◽  
Calcium Currents ◽  
Dorsal Root Ganglion Neurons ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Low Threshold ◽  
Ganglion Neurons

Bouskila, Yona and Hugh Bostock. Modulation of voltage-activated calcium currents by mechanical stimulation in rat sensory neurons. J. Neurophysiol. 80: 1647–1652, 1998. We examined the effects of mechanical stress, induced by a stream of bath solution, on evoked action potentials, electrical excitability, and Ca2+ currents in rat dorsal root ganglion neurons in culture with the use of the whole cell patch-clamp technique. Action-potential duration was altered reversibly by flow in 39% of the 51 neurons tested, but membrane potential and excitability were unaffected. The flow-induced increases and decreases in action-potential duration were consistent with the different effects of flow on two types of Ca2+ channel, determined by voltage-clamp recordings of Ba2+ currents. Current through ω-conotoxin–sensitive (N-type) Ca2+ channels increased by an estimated 74% with flow, corresponding to 23% increase in the total high voltage–activated current, whereas current through low-threshold voltage-activated (T-type) channels decreased by 14%. We conclude that modulation of voltage-activated Ca2+ currents constitutes a route by which mechanical events can regulate Ca2+ influx in sensory neurons.

Mechanosensitive currents in putative aortic baroreceptor neurons in vitro

1995 ◽  
Vol 73 (5) ◽  
pp. 2094-2098 ◽  
Author(s):  
J. T. Cunningham ◽  
R. E. Wachtel ◽  
F. M. Abboud
Keyword(s):  
Reversal Potential ◽  
Tumor Cell Line ◽  
Mouse Tumor ◽  
Mechanosensitive Channels ◽  
Inward Current ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channels ◽  
Whole Cell Patch Clamp ◽  
Nodose Ganglia

1. Whole cell patch-clamp experiments were conducted to determine whether rat aortic baroreceptor neurons contain mechano-sensitive conductances. 2. Putative aortic baroreceptor neurons in the nodose ganglia were identified by injecting DiI onto the adventitia of the aortic arch. Nodose ganglia neurons were dissociated after > or = 1 wk. A fluorescein-conjugated tetanus toxin fragment was used to confirm that the cells labeled with DiI in culture were neurons. 3. Hypoosmotic stretch significantly increased the conductance of DiI-labeled neurons (n = 19). The reversal potential of the response was -11 +/- 1 (SE) mV. 4. In experiments on unlabeled neurons, only 7 of 13 cells showed increases in conductance. BC3H1 cells, a mouse tumor cell line, showed no changes in conductance. 5. Gadolinium (20 microM), a putative blocker of mechanosensitive channels, prevented the increase in conductance produced by hypoosmolality in seven of seven labeled cells. Equimolar concentrations of lanthanum (n = 6) and omega-conotoxin GVIA (1 microM, n = 4), which block voltage-gated calcium channels, failed to significantly affect the inward current.

Comparison of a Ca(2+)-gated conductance and a second-messenger-gated conductance in rat olfactory neurons

2000 ◽  
Vol 203 (3) ◽  
pp. 567-573
Author(s):  
Y. Okada ◽  
R. Fujiyama ◽  
T. Miyamoto ◽  
T. Sato
Keyword(s):  
Outward Current ◽  
Patch Clamp Technique ◽  
External Application ◽  
Olfactory Neurons ◽  
Internal Solution ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Cation Conductance ◽  
Intracellular Ca ◽  
Sustained Outward Current

The effect of a rise in intracellular Ca(2+) concentration was analyzed in isolated rat olfactory neurons using a whole-cell patch-clamp technique. Intracellular dialysis of 1 mmol l(−)(1) Ca(2+) in a standard-K(+), low-Cl(−) internal solution (E(Cl)=−69 mV) from the patch pipette into the olfactory neurons induced a sustained outward current of 49+/−5 pA (N=13) at −50 mV in all the cells examined. The outward currents were inhibited by external application of 100 micromol l(−)(1) 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). External application of a Ca(2+) ionophore, 3 micromol l(−)(1) ionomycin, induced an inward current in three of eight cells whose voltages were clamped using the gramicidin-perforated technique, but ionomycin elicited an outward current in the other five cells, suggesting that natural intracellular Cl(−) concentration in the olfactory neurons was heterogeneous. While intracellular dialysis of 50 micromol l(−)(1) inositol 1,4,5-trisphosphate (1,4,5-InsP(3)) in the standard-K(+), low-Cl(−) internal solution induced the NPPB-sensitive outward current in 31 % of cells, and 500 micromol l(−)(1) cAMP induced it in 21 % of cells, a large proportion of the cells displayed an inward current in response to 1,4,5-InsP(3) and cAMP. The results suggest that 1,4,5-InsP(3) and cAMP can elicit Ca(2+)-dependent Cl(−) conductance and Ca(2+)-independent cation conductance in rat olfactory neurons.

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