scholarly journals Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A

2016 ◽  
Vol 148 (5) ◽  
pp. 375-392 ◽  
Author(s):  
Novandy K. Lim ◽  
Andy K.M. Lam ◽  
Raimund Dutzler
Keyword(s):  
Ion Selectivity ◽  
Functional Independence ◽  
Structural Features ◽  
Ion Conduction ◽  
Functional Behavior ◽  
Cl Channels ◽  
Patch Clamp Electrophysiology ◽  
Common Architecture ◽  
Functional Breadth ◽  
Pore Lining

The TMEM16 proteins constitute a family of membrane proteins with unusual functional breadth, including lipid scramblases and Cl− channels. Members of both these branches are activated by Ca2+, acting from the intracellular side, and probably share a common architecture, which was defined in the recent structure of the lipid scramblase nhTMEM16. The structural features of subunits and the arrangement of Ca2+-binding sites in nhTMEM16 suggest that the dimeric protein harbors two locations for catalysis that are independent with respect to both activation and lipid conduction. Here, we ask whether a similar independence is observed in the Ca2+-activated Cl− channel TMEM16A. For this purpose, we generated concatenated constructs containing subunits with distinct activation and permeation properties. Our biochemical investigations demonstrate the integrity of concatemers after solubilization and purification. During investigation by patch-clamp electrophysiology, the functional behavior of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. This resemblance extends to ion selectivity, conductance, and the concentration and voltage dependence of channel activation by Ca2+. Constructs combining subunits with different potencies for Ca2+ show a biphasic activation curve that can be described as a linear combination of the properties of its constituents. The functional independence is further supported by mutation of a putative pore-lining residue that changes the conduction properties of the mutated subunit. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca2+ binding to sites that are embedded within the transmembrane part of each subunit.

scholarly journals Calmodulin Contributes to Gating Control in Olfactory Calcium-activated Chloride Channels

2006 ◽  
Vol 127 (6) ◽  
pp. 737-748 ◽  
Author(s):  
Hiroshi Kaneko ◽  
Frank Möhrlen ◽  
Stephan Frings
Keyword(s):  
Sensory Neurons ◽  
Signal Amplification ◽  
Single Channel ◽  
Feedback Inhibition ◽  
Ion Selectivity ◽  
Sensory Information ◽  
Activation Mechanism ◽  
Ca Channels ◽  
Cl Channels ◽  
Ca Sensitivity

In sensory neurons of the peripheral nervous system, receptor potentials can be amplified by depolarizing Cl currents. In mammalian olfactory sensory neurons (OSNs), this anion-based signal amplification results from the sequential activation of two distinct types of transduction channels: cAMP-gated Ca channels and Ca-activated Cl channels. The Cl current increases the initial receptor current about 10-fold and leads to the excitation of the neuron. Here we examine the activation mechanism of the Ca-dependent Cl channel. We focus on calmodulin, which is known to mediate Ca effects on various ion channels. We show that the cell line Odora, which is derived from OSN precursor cells in the rat olfactory epithelium, expresses Ca-activated Cl channels. Single-channel conductance, ion selectivity, voltage dependence, sensitivity to niflumic acid, and Ca sensitivity match between Odora channels and OSN channels. Transfection of Odora cells with CaM mutants reduces the Ca sensitivity of the Cl channels. This result points to the participation of calmodulin in the gating process of Ca-ativated Cl channels, and helps to understand how signal amplification works in the olfactory sensory cilia. Calmodulin was previously shown to mediate feedback inhibition of cAMP-synthesis and of the cAMP-gated Ca channels in OSNs. Our results suggest that calmodulin may also be instrumental in the generation of the excitatory Cl current. It appears to play a pivotal role in the peripheral signal processing of olfactory sensory information. Moreover, recent results from other peripheral neurons, as well as from smooth muscle cells, indicate that the calmodulin-controlled, anion-based signal amplification operates in various cell types where it converts Ca signals into membrane depolarization.

Cl- channels of the gastric parietal cell that are active at low pH

1993 ◽  
Vol 264 (6) ◽  
pp. C1609-C1618 ◽  
Author(s):  
J. Cuppoletti ◽  
A. M. Baker ◽  
D. H. Malinowska
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Parietal Cell ◽  
Asymmetric Reduction ◽  
Ion Selectivity ◽  
Channel Activity ◽  
Open Probability ◽  
Gastric Parietal Cell ◽  
Cl Channel ◽  
Cl Channels

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+-activated Cl− channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection

2003 ◽  
Vol 94 (1) ◽  
pp. 301-313 ◽  
Author(s):  
Tom Karkanis ◽  
Ling DeYoung ◽  
Gerald B. Brock ◽  
Stephen M. Sims
Keyword(s):  
Smooth Muscle ◽  
Penile Erection ◽  
Corpus Cavernosum ◽  
Outward Current ◽  
Channel Blockers ◽  
Inward Current ◽  
Inward Currents ◽  
Cl Channels ◽  
Patch Clamp Electrophysiology

Little is known of the excitatory mechanisms that contribute to the tonic contraction of the corpus cavernosum smooth muscle in the flaccid state. We used patch-clamp electrophysiology to investigate a previously unidentified inward current in freshly isolated rat and human corporal myocytes. Phenylephrine (PE) contracted cells and activated whole cell currents. Outward current was identified as large-conductance Ca2+-activated K+ current. The inward current elicited by PE was dependent on the Cl− gradient and was inhibited by niflumic acid, indicative of a Ca2+-activated Cl− (ClCa) current. Furthermore, spontaneous transient outward and inward currents (STOCs and STICs, respectively) were identified in both rat and human corporal myocytes and derived from large-conductance Ca2+-activated K+ and ClCa channel activity. STICs and STOCs were inhibited by PE and A-23187, and combined 8-bromoadenosine cAMP and 8-bromoadenosine cGMP decreased their frequency. When studied in vivo, chloride channel blockers transiently increased intracavernosal pressure and prolonged nerve-evoked erections. This report reveals for the first time ClCa current in rat and human corpus cavernosum smooth muscle cells and demonstrates its key functional role in the regulation of penile erection.

scholarly journals TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (8) ◽  
pp. 4266-4273 ◽  
Author(s):  
C H Ko ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Functional Independence ◽  
Structural Features ◽  
Low Ph ◽  
Sequence Identity ◽  
Hydrophilic Region ◽  
Membrane Spanning ◽  
Delta Cells ◽  
Membrane Spanning Domains

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

scholarly journals Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alexandria N Miller ◽  
George Vaisey ◽  
Stephen B Long
Keyword(s):  
Electron Microscopy ◽  
Ion Channels ◽  
Molecular Mechanisms ◽  
Peptide Binding ◽  
Retinal Disease ◽  
Structural Similarity ◽  
Cryo Electron Microscopy ◽  
Cl Channels ◽  
Pore Lining ◽  
Protein Shell

Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel’s principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.

scholarly journals Atomistic basis of opening and conduction in mammalian inward rectifier potassium (Kir2.2) channels

2019 ◽  
Author(s):  
Eva-Maria Zangerl-Plessl ◽  
Sun-Joo Lee ◽  
Grigory Maksaev ◽  
Harald Bernsteiner ◽  
Feifei Ren ◽  
...  
Keyword(s):  
Md Simulations ◽  
Inward Rectifier ◽  
Ion Conduction ◽  
Kir Channel ◽  
Channel Opening ◽  
Spontaneous Wetting ◽  
Pore Lining ◽  
Crossing Point ◽  
Gate Region

Potassium ion conduction through open potassium channels is essential to control of membrane potentials in all cells. To elucidate the open conformation and hence the mechanism of K+ ion conduction in the classical inward rectifier Kir2.2, we introduced a negative charge (G178D) at the crossing point of the inner helix bundle (HBC), the location of ligand-dependent gating. This ‘forced open’ mutation generated channels that were active even in the complete absence of phosphoinositol-4,5-bisphosphate (PIP2), an otherwise essential ligand for Kir channel opening. Crystal structures were obtained at a resolution of 3.6 Å without PIP2 bound, or 2.8 Å in complex with PIP2. The latter revealed a slight widening at the HBC, through backbone movement. Molecular dynamics (MD) simulations showed that subsequent spontaneous wetting of the pore through the HBC gate region allowed K+ ion movement across the HBC and conduction through the channel. Further simulations reveal atomistic details of the opening process and highlight the role of pore lining acidic residues in K+ conduction through Kir2 channels.

scholarly journals The structure of the Pro-domain of mouse proNGF in contact with the NGF domain

2018 ◽  
Author(s):  
Robert Yan ◽  
Havva Yalinca ◽  
Francesca Paoletti ◽  
Francesco Gobbo ◽  
Laura Marchetti ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Functional Characterization ◽  
Functional Independence ◽  
Structural Features ◽  
Compact Structure ◽  
Hybrid Strategy ◽  
Growth And Survival ◽  
Functional Roles ◽  
Growth Cone Collapse ◽  
Protease Cleavage

AbstractNerve Growth Factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation, maintenance, growth and survival of target neurons. Expressed as a proNGF precursor, NGF is then matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct cellular partners which account for different functional roles. While the structure of mature NGF is available, little is known about the structure of the pro-domain within the context of proNGF because the dynamical and structural features of the protein have so far prevented its structure determination. We have exploited a new hybrid strategy based on nuclear magnetic resonance and modelling validated by small angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but has a clear tertiary structure propensity and forms transient tertiary intramolecular contacts. It is also able to interact, albeit weakly, with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Based on paramagnetic relaxation enhancement data and advanced molecular modelling, we have then reconstructed the overall properties of the pro-domain in the context of proNGF and showed that it has a compact structure. Our data represent an important step towards the structural and functional characterization of the properties of proNGF and its pro-domain.

TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (8) ◽  
pp. 4266-4273 ◽  
Author(s):  
C H Ko ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Functional Independence ◽  
Structural Features ◽  
Low Ph ◽  
Sequence Identity ◽  
Hydrophilic Region ◽  
Membrane Spanning ◽  
Delta Cells ◽  
Membrane Spanning Domains

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

Deep Understanding of Technical Documents: Part I. Diagrams Structural-functional Modeling

2021 ◽  
Vol 30 (03) ◽  
pp. 2150015
Author(s):  
N. G. Bourbakis ◽  
G. Rematska ◽  
S. Mertoguno
Keyword(s):  
Formal Language ◽  
Block Diagram ◽  
Deep Understanding ◽  
Structural Features ◽  
Stochastic Petri Nets ◽  
Functional Modeling ◽  
Natural Language Text ◽  
Functional Behavior ◽  
Technical Documents ◽  
Language Text

The automatic deep understanding of technical documents is a privilege only to humans so far, since it requires knowledge coming from many different modalities, like text, diagrams, formulas, tables, graphics, pictures, etc. Thus, in response to this very large and complex challenge, this paper investigates the synergistic association of only two modalities, the diagrams as main modality and natural language text as an assistive one in an effort to combine them together for deeper understanding of technical documents. In particular, it presents the formal modelling of a hybrid methodology capable to automatically extract the structural and functional behavior of a system described in a technical document without the use of original code. By system here we mean the block diagram(s) of a system. The methodology presented here is based on a formal language, called Synergy, to efficiently represent and synthesize the structural features of the system, and convert them into a Stochastic Petri-nets (SPN) model as for expressing the functional behavior of the understudy system. The overall methodology will contribute to an automatic deep understanding of technical documents (TD) without the main involvement of human users.

cAMP-activated chloride conductance in the colonic cell line, Caco-2

1992 ◽  
Vol 262 (1) ◽  
pp. C251-C256 ◽  
Author(s):  
C. E. Bear ◽  
E. F. Reyes
Keyword(s):  
Reversal Potential ◽  
Channel Activity ◽  
Cell Potential ◽  
Linear Current ◽  
Current Voltage ◽  
Patch Pipette ◽  
Cl Channels ◽  
Patch Clamp Electrophysiology ◽  
Best Fit ◽  
Linear Regressions

In this study we investigated the properties of adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- efflux in Caco-2 monolayers by measuring 125I efflux rates from preloaded cells and using patch-clamp electrophysiology. The addition of a cocktail containing 100 microM dibutyryl cAMP (DBcAMP), 10 microM forskolin, and 1 mM 3-isobutyl-1-methylxanthine caused a significant (P less than 0.05) increase in the rate of 125I efflux. Dissipation of cell potential by adding valinomycin (4.5 microM) with 135 mM extracellular KCl reduced the cAMP-evoked 125I efflux. These results suggest that cAMP-stimulated anion efflux occurs through a conductive pore or channel. Whole cell currents evoked with DBcAMP or forskolin were anion selective, PCl greater than PI greater than Pgluconate, and exhibited a linear current-voltage (I-V) relationship. Currents evoked with depolarizing or hyperpolarizing voltage steps showed no evidence of time-dependent activation or inactivation. Single Cl- channels were stimulated in cell-attached patches after treatment with cAMP. Onset of channel activity occurred after 20-30s of cAMP treatment, and the response was long lasting. The I-V relationship for the channel activated in cell-attached patches by cAMP was best fit using two linear regressions. The slope conductance of the channel was 3.2 +/- 0.6 and 7.4 +/- 0.3 pS at hyperpolarizing and depolarizing potentials, respectively. Substitution of 140 mM NaCl with 70 mM NaCl in the patch pipette resulted in a positive shift in reversal potential, indicating that the channel is anion selective.(ABSTRACT TRUNCATED AT 250 WORDS)

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