scholarly journals Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

2015 ◽  
Vol 145 (4) ◽  
pp. 285-301 ◽  
Author(s):  
Asma Amjad ◽  
Andres Hernandez-Clavijo ◽  
Simone Pifferi ◽  
Devendra Kumar Maurya ◽  
Anna Boccaccio ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Functional Characterization ◽  
Vomeronasal Organ ◽  
Conditional Knockout ◽  
Channel Blockers ◽  
Whole Cell ◽  
Knock Out ◽  
Cl Channels ◽  
Inside Out ◽  
Vomeronasal Sensory Neurons

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca2+ concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca2+ in isolated mouse vomeronasal sensory neurons in the absence of intracellular K+. In whole-cell recordings, the average current in 1.5 µM Ca2+ and symmetrical Cl− was −382 pA at −100 mV. Ion substitution experiments and partial blockade by commonly used Cl− channel blockers indicated that Ca2+ activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca2+-activated Cl− channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca2+-activated Cl− channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre–loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca2+-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca2+-activated Cl− currents in mouse vomeronasal sensory neurons.

scholarly journals Calcium-activated chloride current amplifies the response to urine in mouse vomeronasal sensory neurons

2009 ◽  
Vol 135 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chun Yang ◽  
Rona J. Delay
Keyword(s):  
Sensory Neurons ◽  
Detection System ◽  
Reversal Potential ◽  
Vomeronasal Organ ◽  
Channel Blockers ◽  
Cl Channel ◽  
Chloride Accumulation ◽  
Perforated Patch Clamp ◽  
Whole Cell Recordings ◽  
Vomeronasal Sensory Neurons

The vomeronasal organ (VNO) is an odor detection system that mediates many pheromone-sensitive behaviors. Vomeronasal sensory neurons (VSNs), located in the VNO, are the initial site of interaction with odors/pheromones. However, how an individual VSN transduces chemical signals into electrical signals is still unresolved. Here, we show that a Ca2+-activated Cl− current contributes ∼80% of the response to urine in mouse VSNs. Using perforated patch clamp recordings with gramicidin, which leaves intracellular chloride undisrupted, we found that the urine-induced inward current (Vhold = −80 mV) was decreased in the presence of chloride channel blockers. This was confirmed using whole cell recordings and altering extracellular chloride to shift the reversal potential. Further, the urine-induced currents were eliminated when both extracellular Ca2+ and Na+ were removed. Using inside-out patches from dendritic tips, we recorded Ca2+-activated Cl− channel activity. Several candidates for this Ca2+-activated Cl− channel were detected in VNO by reverse transcription–polymerase chain reaction. In addition, a chloride cotransporter, Na+-K+-2Cl− isoform 1, was detected and found to mediate much of the chloride accumulation in VSNs. Collectively, our data demonstrate that chloride acts as a major amplifier for signal transduction in mouse VSNs. This amplification would increase the responsiveness to pheromones or odorants.

scholarly journals Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

2015 ◽  
Vol 212 (5) ◽  
pp. 2125OIA23
Author(s):  
Asma Amjad ◽  
Andres Hernandez-Clavijo ◽  
Simone Pifferi ◽  
Devendra Kumar Maurya ◽  
Anna Boccaccio ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Conditional Knockout ◽  
Chloride Current ◽  
Vomeronasal Sensory Neurons

scholarly journals Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

cAMP-independent regulation of CFTR by the actin cytoskeleton

1995 ◽  
Vol 268 (6) ◽  
pp. C1552-C1561 ◽  
Author(s):  
A. G. Prat ◽  
Y. F. Xiao ◽  
D. A. Ausiello ◽  
H. F. Cantiello
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Mammary Adenocarcinoma ◽  
Actin Binding ◽  
Whole Cell ◽  
Deoxyribonuclease I ◽  
Epithelial Na Channels ◽  
Cytosolic Side ◽  
Cl Channels ◽  
Inside Out

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.

cAMP-activated Cl- channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland

1995 ◽  
Vol 268 (1) ◽  
pp. C70-C79 ◽  
Author(s):  
D. C. Devor ◽  
J. N. Forrest ◽  
W. K. Suggs ◽  
R. A. Frizzell
Keyword(s):  
Single Channel ◽  
Primary Cultures ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Spiny Dogfish ◽  
Open Probability ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Cl Channels ◽  
Inside Out

Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.

Similarity of A3-adenosine and swelling-activated Cl− channels in nonpigmented ciliary epithelial cells

2000 ◽  
Vol 279 (2) ◽  
pp. C440-C451 ◽  
Author(s):  
David A. Carré ◽  
Claire H. Mitchell ◽  
Kim Peterson-Yantorno ◽  
Miguel Coca-Prados ◽  
Mortimer M. Civan
Keyword(s):  
Reversal Potential ◽  
Partial Replacement ◽  
Channel Blockers ◽  
Patch Clamping ◽  
Whole Cell ◽  
Selective Agonist ◽  
Outward Currents ◽  
Cl Channels ◽  
Adenosine Agonists ◽  
Nonpigmented Ciliary Epithelial

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl− transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A3-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)- N-methyl-β-d-ribofuranuronamide (IB-MECA) stimulated currents ( I IB-MECA) by ∼90% at +80 mV. Partial replacement of external Cl−with aspartate reduced outward currents and shifted the reversal potential ( V rev) from −23 ± 2 mV to −0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl− replacement with aspartate and NO3 −, and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents ( I Swell). Partial cyclamate substitution for external Cl− inhibited inward and outward currents of both I IB-MECA and I Swell. Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A3-subtype adenosine agonists and swelling activate a common population of Cl− channels.

Adenosine stimulates Cl− channels of nonpigmented ciliary epithelial cells

1997 ◽  
Vol 273 (4) ◽  
pp. C1354-C1361 ◽  
Author(s):  
David A. Carré ◽  
Claire H. Mitchell ◽  
Kim Peterson-Yantorno ◽  
Miguel Coca-Prados ◽  
Mortimer M. Civan
Keyword(s):  
Epithelial Cells ◽  
Adenosine Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ciliary Epithelium ◽  
Channel Blockers ◽  
Whole Cell ◽  
Cl Channels ◽  
Opposing Effects ◽  
Nonpigmented Ciliary Epithelial

Ciliary epithelial cells possess multiple purinergic receptors, and occupancy of A1 and A2 adenosine receptors is associated with opposing effects on intraocular pressure. Aqueous adenosine produced increases in short-circuit current across rabbit ciliary epithelium, blocked by removing Cl− and enhanced by aqueous Ba2+. Adenosine’s actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells. With gramicidin present, adenosine (≥3 μM) triggered isosmotic shrinkage of the human NPE cells, which was inhibited by the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 μM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (≥1 μM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl− dependent, and reversibly inhibited by NPPB. Adenosine (≥10 μM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl− secretion in mammalian NPE cells.

cAMP-activated maxi-Cl− channels in native bovine pigmented ciliary epithelial cells

2004 ◽  
Vol 287 (4) ◽  
pp. C1003-C1011 ◽  
Author(s):  
Chi-Wai Do ◽  
Kim Peterson-Yantorno ◽  
Claire H. Mitchell ◽  
Mortimer M. Civan
Keyword(s):  
Aqueous Humor ◽  
Ciliary Epithelium ◽  
Whole Cell ◽  
Channel Inactivation ◽  
Cytoplasmic Surface ◽  
Unitary Conductance ◽  
Cell Layers ◽  
Cl Channels ◽  
Excised Patches ◽  
Inside Out

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl− enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl− channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl− channels with a unitary conductance of 272 ± 2 pS ( n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl− channels. cAMP also activated the maxi-Cl− channels in outside-out patches. In both cases, maxi-Cl− channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl− concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl− concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl− channels of native PE cells that contribute to Cl− release particularly from Cl−-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl− reabsorption into the ciliary stroma.

scholarly journals The transcription factor Tfap2e/AP-2ε plays a pivotal role in maintaining the identity of basal vomeronasal sensory neurons

2018 ◽  
Author(s):  
Jennifer M Lin ◽  
Ed Zandro M Taroc ◽  
Jesus A Frias ◽  
Aparna Prasad ◽  
Allison N Catizone ◽  
...  
Keyword(s):  
Transcription Factor ◽  
G Protein ◽  
Sensory Neurons ◽  
Control Cell ◽  
Neuronal Cell ◽  
Vomeronasal Organ ◽  
Cell Types ◽  
Protein Subunit ◽  
Cell Type Specific ◽  
Vomeronasal Sensory Neurons

The identity of individual neuronal cell types is defined by the expression of specific combinations of transcriptional regulators that control cell type-specific genetic programs. The epithelium of the vomeronasal organ of mice contains two major types of vomeronasal sensory neurons (VSNs): 1) the apical VSNs which express vomeronasal 1 receptors (V1r) and the G-protein subunit Gαi2 and; 2) the basal VSNs which express vomeronasal 2 receptors (V2r) and the G-protein subunit Gαo. Both cell types originate from a common pool of progenitors and eventually acquire apical or basal identity through largely unknown mechanisms. The transcription factor AP-2ε, encoded by the Tfap2e gene, plays a role in controlling the development of GABAergic interneurons in the main and accessory olfactory bulb (AOB), moreover AP-2ε has been previously described to be expressed in the VSNs. Here we show that AP-2ε is expressed in postmitotic VSNs after they commit to the basal differentiation program. Loss of AP-2ε function resulted in reduced number of basal VSNs and in an increased number of neurons expressing markers of the apical lineage. Our work suggests that AP-2ε, which is expressed in late phases of differentiation, is not needed to initiate the apical-basal differentiation dichotomy but for maintaining the basal VSNs' identity by preventing the expression of apical genes. Moreover, our data suggest that differentiated VSNs of mice retain a notable level of plasticity.

scholarly journals Smad4 signaling establishes the somatosensory map of basal vomeronasal sensory neurons

2019 ◽  
Author(s):  
Ankana S. Naik ◽  
Jennifer M. Lin ◽  
Ed Zandro M. Taroc ◽  
Raghu R. Katreddi ◽  
Jesus A. Frias ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Critical Role ◽  
Vomeronasal Organ ◽  
Bmp Signaling ◽  
Loss Of Function ◽  
Neuronal Populations ◽  
Innervation Patterns ◽  
Circuit Formation ◽  
Accessory Olfactory System ◽  
Vomeronasal Sensory Neurons

SummaryThe accessory olfactory system is a unique model that can give insights on how the neurons can establish and maintain their identity, and connectivity. The vomeronasal organ (VNO) contains two distinct populations of vomeronasal sensory neurons (VSNs) each with specific innervation patterns to the accessory olfactory bulb (AOB). Though morphogenic signals are critical in defining various neuronal populations, the morphogenic signaling profiles that influence each VSN population remains unknown. Here, we found a pronounced BMP signaling gradient within the basal VSNs. By generating Smad4 conditional mutants, we disrupted canonical TGF-β/BMP signaling in maturing basal VSNs and in all mature VSNs. We show that Smad4 loss-of-function in immature basal neurons leads to a progressive loss of basal VSNs, reduced activation of the remnant basal VSNs, and aberrant glomeruli formation in posterior AOB. However, Smad4 ablation in all mature VSNs does not affect neuronal activity nor survival but causes aberrant glomeruli formation only in the posterior AOB. Our study reveals that Smad4 signaling plays a critical role in mediating development, function, and circuit formation of basal VSNs.

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