scholarly journals Release and Elementary Mechanisms of Nitric Oxide in Hair Cells

2010 ◽  
Vol 103 (5) ◽  
pp. 2494-2505 ◽  
Author(s):  
Ping Lv ◽  
Adrian Rodriguez-Contreras ◽  
Hyo Jeong Kim ◽  
Jun Zhu ◽  
Dongguang Wei ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inner Ear ◽  
Hair Cells ◽  
Single Channel ◽  
Cell Types ◽  
Cellular Mechanism ◽  
Open Probability ◽  
Potential Oscillations ◽  
Efferent Nerve ◽  
Membrane Oscillations

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency–current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca2+ current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca2+ channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca2+-activated K+ currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.

Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels

1998 ◽  
Vol 274 (4) ◽  
pp. L475-L484 ◽  
Author(s):  
Lucky Jain ◽  
Xi-Juan Chen ◽  
Lou Ann Brown ◽  
Douglas C. Eaton
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Cation Channel ◽  
Alveolar Type ◽  
Apical Surface ◽  
Type Ii ◽  
Patch Clamp Technique ◽  
Open Probability

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( P o), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the P o by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; P o, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; −42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( P o, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

2005 ◽  
Vol 289 (3) ◽  
pp. C717-C726 ◽  
Author(s):  
My N. Helms ◽  
Ling Yu ◽  
Bela Malik ◽  
Dean J. Kleinhenz ◽  
C. Michael Hart ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Single Channel ◽  
Short Circuit ◽  
Primary Cultures ◽  
Alveolar Epithelium ◽  
Alveolar Type ◽  
No Production ◽  
Open Probability ◽  
No Donor

Several studies have shown that nitric oxide (NO) inhibits Na+ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na+ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 μM dexamethasone and 10% bovine serum. After 1.5 μM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound ( P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability ( Po) from 0.186 ± 0.043 to 0.045 ± 0.009 ( n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na+ transport is by altering iNOS production of NO.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

scholarly journals Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death

1999 ◽  
Vol 277 (4) ◽  
pp. C673-C683 ◽  
Author(s):  
Muyiwa Gbadegesin ◽  
Stefano Vicini ◽  
Sandra J. Hewett ◽  
David A. Wink ◽  
Michael Espey ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Steady State ◽  
Nmda Receptor ◽  
Single Channel ◽  
Receptor Activation ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Nmda Channel

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-d-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that ( Z)-1-[ N-(3-ammoniopropyl)- N-( n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.

Cell fate choices and the expression of Notch, Delta and Serrate homologues in the chick inner ear: parallels with Drosophila sense-organ development

Development ◽  
1998 ◽  
Vol 125 (23) ◽  
pp. 4645-4654 ◽  
Author(s):  
J. Adam ◽  
A. Myat ◽  
I. Le Roux ◽  
M. Eddison ◽  
D. Henrique ◽  
...  
Keyword(s):  
Inner Ear ◽  
Cell Fate ◽  
Hair Cells ◽  
Early Stage ◽  
Sense Organ ◽  
Cell Types ◽  
Developmental Pathways ◽  
Supporting Cells ◽  
Notch Ligand ◽  
Developmental Program

The sensory patches in the vertebrate inner ear are similar in function to the mechanosensory bristles of a fly, and consist of a similar set of cell types. If they are truly homologous structures, they should also develop by similar mechanisms. We examine the genesis of the neurons, hair cells and supporting cells that form the sensory patches in the inner ear of the chick. These all arise from the otic epithelium, and are produced normally even in otic epithelium cultured in isolation, confirming that their production is governed by mechanisms intrinsic to the epithelium. First, the neuronal sublineage becomes separate from the epithelial: between E2 and E3.5, neuroblasts delaminate from the otocyst. The neuroblasts then give rise to a mixture of neurons and neuroblasts, while the sensory epithelial cells diversify to form a mixture of hair cells and supporting cells. The epithelial patches where this occurs are marked from an early stage by uniform and maintained expression of the Notch ligand Serrate1. The Notch ligand Delta1 is also expressed, but transiently and in scattered cells: it is seen both early, during neuroblast segregation, where it appears to be in the nascent neuroblasts, and again later, in the ganglion and in differentiating sensory patches, where it appears to be in the nascent hair cells, disappearing as they mature. Delta-Notch-mediated lateral inhibition may thus act at each developmental branchpoint to drive neighbouring cells along different developmental pathways. Our findings indicate that the sensory patches of the vertebrate inner ear and the sensory bristles of a fly are generated by minor variations of the same basic developmental program, in which cell diversification driven by Delta-Notch and/or Serrate-Notch signalling plays a central part.

scholarly journals Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation

2009 ◽  
Vol 297 (1) ◽  
pp. C75-C85 ◽  
Author(s):  
Mingjie Tong ◽  
R. Keith Duncan
Keyword(s):  
Hair Cells ◽  
Molecular Mechanisms ◽  
Single Channel ◽  
Frequency Tuning ◽  
Low Frequency ◽  
Bk Channels ◽  
Bk Channel ◽  
Basilar Papilla ◽  
Channel Kinetics ◽  
Open Probability

Large-conductance, Ca2+-activated, and voltage-gated potassium channels (BK, BKCa, or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary β1 have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the β-subunit, was used to test for the functional presence of β1. The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of β1 and that the level of activation would exhibit a tonotopic gradient following the expression profile of β1. Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary β-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla.

Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells

2007 ◽  
Vol 293 (5) ◽  
pp. F1666-F1677 ◽  
Author(s):  
Ling Yu ◽  
Hui-Fang Bao ◽  
Julie L. Self ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Single Channel ◽  
Na Channel ◽  
Dependent Manner ◽  
Channel Measurements ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
A Cell

Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2−) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 μM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2−(with the O2−scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 ± 0.03 to 0.03 ± 0.01. We also found that increasing endogenous O2−in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 ± 0.05) after superoxide dismutase and 1.5 μM NO coincubation (0.21 ± 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2−by ∼30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2−alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.

scholarly journals Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Tomoko Hyakumura ◽  
Stuart McDougall ◽  
Sue Finch ◽  
Karina Needham ◽  
Mirella Dottori ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Inner Ear ◽  
Hair Cells ◽  
Cochlear Nucleus ◽  
Cell Types ◽  
Quantitative Evidence ◽  
Brainstem Slice

Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.

scholarly journals Alternative Splicing of Cdh23 Exon 68 Is Regulated by RBM24, RBM38, and PTBP1

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Nana Li ◽  
Haibo Du ◽  
Rui Ren ◽  
Yanfei Wang ◽  
Zhigang Xu
Keyword(s):  
Hearing Loss ◽  
Alternative Splicing ◽  
Inner Ear ◽  
Hair Cells ◽  
Cell Types ◽  
Splicing Factors ◽  
Mechanoelectrical Transduction ◽  
Eukaryotic Proteins ◽  
A Cell ◽  
Cadherin 23

Alternative splicing plays a pivotal role in modulating the function of eukaryotic proteins. In the inner ear, many genes undergo alternative splicing, and errors in this process lead to hearing loss. Cadherin 23 (CDH23) forms part of the so-called tip links, which are indispensable for mechanoelectrical transduction (MET) in the hair cells. Cdh23 gene contains 69 exons, and exon 68 is subjected to alternative splicing. Exon 68 of the Cdh23 gene is spliced into its mRNA only in a few cell types including hair cells. The mechanism responsible for the alternative splicing of Cdh23 exon 68 remains elusive. In the present work, we performed a cell-based screening to look for splicing factors that regulate the splicing of Cdh23 exon 68. RBM24 and RBM38 were identified to enhance the inclusion of Cdh23 exon 68. The splicing of Cdh23 exon 68 is affected in Rbm24 knockdown or knockout cells. Moreover, we also found that PTBP1 inhibits the inclusion of Cdh23 exon 68. Taken together, we show here that alternative splicing of Cdh23 exon 68 is regulated by RBM24, RBM38, and PTBP1.

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