Full Open State Research Articles

Research and development of innovative bidirectional control plate valve for reciprocating compressor

Currently, the reciprocating compressor valve operates on a single working principle, where the valve structure relies on the combined force of its own elasticity and gas thrust to drive the movement of the valve disc, and continues to open and close in the way of impact. Due to the defects of the conventional plate valve, such as delayed opening, premature closing, and insufficient full opening time, in combination with the self-acting valve structure and constant volume cylinder where artificial adjustment of compressed air volume is not feasible. Therefore an innovative bidirectional control plate valve has been developed in this study, which employs a linear motor to regulate the valve disc and eliminates structural components such as springs, buffer discs, and valve lift guard. Throughout the entire opening and closing cycle, precise control of the valve disc enables swift operation with prolonged full open state, minimal resistance loss, and absence of impact, vibration or rebound. Its ideal movement mode can directly and accurately achieve stepless capacity regulation. The primary suction valve of the industrial 3LW-5-8 reciprocating compressor is taken as the research subject in this paper. Fluent software is utilized to design the structural parameters of an innovative plate valve, and its feasibility and effectiveness are validated through tests conducted on an automated multi-functional experiment table and an L-type reciprocating compressor. The results demonstrate that the new valve exhibits a 21.52% increase in effective flow area compared to the conventional plate valve, along with a 3.02% increase in exhaust volume and a potential reduction of 2.32% in power consumption when replaced one of the four. Moreover, the innovative bidirectional control plate valve demonstrates precise control over high-frequency motion of the valve disc, effectively addressing inherent limitations of traditional plate valves and exhibiting promising application prospects.

Analysis and Treatment of Abnormal Vibration of Unit 2 in a Power Plant

The Unit 2 in a power plant was first started in 2016 after the flow through transformation, the vibration basically reached the excellent standard in the single valve state with the load running. In the sequence valve state, the high and intermediate pressure rotor occurred obvious steam induced vibration fault at high load. By reducing the main steam pressure properly, the No. 2 regulating valve is in full open state as soon as possible, thus reducing the steam exciting force of the high and intermediate pressure rotor, the vibration fluctuation value of the unit is obviously reduced at high load, and the phenomenon of steam induced vibration is effectively suppressed, and the unit can be safely carried out for a long time.

Dysregulated Zn2+ homeostasis impairs cardiac type-2 ryanodine receptor and mitsugumin 23 functions, leading to sarcoplasmic reticulum Ca2+ leakage

Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.

Subconductance Gating and Voltage Sensitivity of Sarcoplasmic Reticulum K+ Channels: A Modeling Approach

Sarcoplasmic reticulum (SR) K+ channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K+ channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K+ channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K+ channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K+ channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (≤0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K+ flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.

Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.

Ligand induced change of β2adrenergic receptor from active to inactive conformation and its implication for the closed/open state of the water channel: insight from molecular dynamics simulation, free energy calculation and Markov state model analysis

The reported crystal structures of β2 adrenergic receptor (β2AR) reveal that the open and closed states of the water channel are correlated with the inactive and active conformations of β2AR. However, more details about the process by which the water channel states are affected by the active to inactive conformational change of β2AR remain illusive. In this work, molecular dynamics simulations are performed to study the dynamical inactive and active conformational change of β2AR induced by inverse agonist ICI 118,551. Markov state model analysis and free energy calculation are employed to explore the open and close states of the water channel. The simulation results show that inverse agonist ICI 118,551 can induce water channel opening during the conformational transition of β2AR. Markov state model (MSM) analysis proves that the energy contour can be divided into seven states. States S1, S2 and S5, which represent the active conformation of β2AR, show that the water channel is in the closed state, while states S4 and S6, which correspond to the intermediate state conformation of β2AR, indicate the water channel opens gradually. State S7, which represents the inactive structure of β2AR, corresponds to the full open state of the water channel. The opening mechanism of the water channel is involved in the ligand-induced conformational change of β2AR. These results can provide useful information for understanding the opening mechanism of the water channel and will be useful for the rational design of potent inverse agonists of β2AR.

Mouse CFTR Exhibits Multiple Characteristic Differences from Human CFTR

Currently available murine CF models have failed to replicate CF-like spontaneous obstructive lung disease until challenged with bacteria or LPS. It has been reported that the delF508-mCFTR mutant does not exhibit mistrafficking behavior as seen in delF508-hCFTR, and that mCFTR single channel behavior is distinctly different from hCFTR. We have investigated and compared the channel behavior of mCFTR and hCFTR expressed in Xenopus oocytes using electrophysiology combined with mutagenesis. The results are: (1) GlyH-101 blocked hCFTR with Kd 4.9 µM while GlyH-101 blocked mCFTR with Kd 32 µM at VM=-60 mV. GlyH-101 failed to block R334A and R334C in both hCFTR and mCFTR. (2) GlyH-101 activated mCFTR but not hCFTR in a concentration-dependent manner with Kd 103 µM. GlyH-101 only exhibited the activation effect on R334A-mCFTR. (3) 10 µM VX-770 transiently increased hCFTR ∼30%, while activated mCFTR in a time-dependent manner to > 2 fold. (4) Glibenclamide (Glyb) only inhibited hCFTR (Kd 22 µM at Vm = −120 mV) but also inhibited mCFTR (Kd 18 µM at Vm = −120 mV) and activated mCFTR (Kd 5 µM at Vm = 100 mV). Similar phenomena were seen in both whole cell (TEVC) and inside-out macropatch configuration. (5) Whereas human CFTR opens to the full open state (f) over 98% of the open time, mCFTR occupies the f state 60% of the open time, with the rest spend in the subconductance 1 (s1) or subconductance 2 (s2) at Vm= −100mV. Conclusion: (1) mCFTR channel behavior differs from hCFTR in multiple respects. (2) The differences between mCFTR and hCFTR will provide a tool in identifying the binding sites and mechanism of VX-770 and GlyH-101. (NIH R01-DK 056481).

Chemical Uncoupling the DHPR-RyR1 Complex by Substituted Halogenated Biphenyls and Diphenylethers

Ca2+ release units (CRU) of skeletal muscle have two components necessary for excitation-contraction coupling (ECC); the DHPR and RyR1 that physically interact to engage conformational coupling that involves both orthograde and retrograde regulation of Ca2+ dynamics during ECC and SR Ca2+ leak when the muscle is at rest. We previously identified that 2,2',3,5',6-pentachlorobiphenyl (PCB95) promotes long-lived stability of the full open state of RyR1 channels (Samso et al 2009). Further investigations PCB95 and its 4- and 5-hydroxy (-OH) derivatives toward rabbit skeletal muscle ryanodine receptor (RyR1) were performed using [3H]ryanodine binding and SR Ca2+ flux analyses. 5-OH metabolites have comparable activity to their respective parent in both assays; however, 4-OH derivatives are unable to trigger Ca2+ release from SR microsomes in the presence of Ca2+-ATPase activity. PCB95 and derivatives are investigated using single channel voltage-clamp and primary murine embryonic muscle cells. Like PCB95, 5-OH-PCB95 quickly and persistently increases channel open probability (Po>0.9) by stabilizing the full-open channel state, whereas 4-OH-PCB95 transiently enhances Po. Ca2+ imaging of myotubes show acute exposure to PCB95 (5μM) potentiates ECC and caffeine responses and partially depletes SR Ca2+ stores. Exposure to 5-OH-PCB95 (5μM) increases cytoplasmic Ca2+, leading to ECC failure in 50% of myotubes with the remainder retaining negligible responses. 4-OH-PCB95 neither increases baseline Ca2+ nor causes ECC failure but depresses ECC and caffeine responses by 50%. With longer (3 h) exposure to 300nM PCB95, 5-OH-PCB95, or 4-OH-PCB95 decreases the number of ECC responsive myotubes by 22%, 81%, and 51% compared with control by depleting SR Ca2+ and/or uncoupling ECC. The position of hydroxylation of PCB95 and chemically related diphenylethers differentially influence RyR1 channel gating kinetics, SR Ca2+ leak, and the pattern of ECC impairment, including ECC uncoupling in intact muscle cells. P01 AR52354.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

TRIC-B channels display labile gating: evidence from the TRIC-A knockout mouse model

Sarcoplasmic/endoplasmic reticulum (SR) and nuclear membranes contain two related cation channels named TRIC-A and TRIC-B. In many tissues, both subtypes are co-expressed, making it impossible to distinguish the distinct single-channel properties of each subtype. We therefore incorporated skeletal muscle SR vesicles derived from Tric-a-knockout mice into bilayers in order to characterise the biophysical properties of native TRIC-B without possible misclassification of the channels as TRIC-A, and without potential distortion of functional properties by detergent purification protocols. The native TRIC-B channels were ideally selective for cations. In symmetrical 210 mM K+, the maximum (full) open channel level (199 pS) was equivalent to that observed when wild-type SR vesicles were incorporated into bilayers. Analysis of TRIC-B gating revealed complex and variable behaviour. Four main sub-conductance levels were observed at approximately 80 % (161 pS), 60 % (123 pS), 46 % (93 pS), and 30 % (60 pS) of the full open state. Seventy-five percent of the channels were voltage sensitive with Po being markedly reduced at negative holding potentials. The frequent, rapid transitions between TRIC-B sub-conductance states prevented development of reliable gating models using conventional single-channel analysis. Instead, we used mean-variance plots to highlight key features of TRIC-B gating in a more accurate and visually useful manner. Our study provides the first biophysical characterisation of native TRIC-B channels and indicates that this channel would be suited to provide counter current in response to Ca2+ release from the SR. Further experiments are required to distinguish the distinct functional properties of TRIC-A and TRIC-B and understand their individual but complementary physiological roles.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1251-y) contains supplementary material, which is available to authorized users.

Voltage-Dependent Stochastic Gating Models of TRIC-B Channels

TRIC-A and TRIC-B are two, related, trimeric intracellular cation channels present in sarcoplasmic/endoplasmic reticulum (SR) and are thought to provide counter-current for SR Ca2+-release. TRIC-B knockout mice die immediately after birth demonstrating the importance of this isoform [Yazawa et al., 2007, Nature, 448, 78-82]. To study the distinct single-channel gating behaviour of TRIC-B, we incorporated skeletal muscle light SR from TRIC-A knockout mice into artificial membranes under voltage-clamp conditions in symmetrical 210 mM K-PIPES, pH 7.2. We developed Markov models of TRIC-B gating, with up to 4 distinct sub-conductance states (S1-S4), using both QuB [Qin F., 2004, Biophys J., 86(3), 1488-501] and our own software. Our models incorporate different connectivity schemes to account for the intrinsic variability in gating that was observed between different channels. Despite the variability, some obvious trends emerged. TRIC-B activity was higher at positive than at negative holding potentials. At positive potentials, the majority of channels exhibited long bursts of openings where predominant gating transitions were between the full open state and S1, the largest sub-conductance state. Some channels, however, gated preferentially in sub-states S3 and S4, only visiting the full open state briefly. At negative potentials, channel activity consisted primarily of brief transitions between sub-conductance states. Closed lifetime distributions at positive potentials comprised of fast components (τ ≈ 1 ms), corresponding to brief transitions from the full open state, as well as slower components corresponding to inter-burst intervals. At negative potentials, inter-burst intervals were orders of magnitude longer demonstrating that the frequency of channel opening is heavily dependent on voltage. It will be important to develop comprehensive models of TRIC-B channel gating in order to fully understand the role of this important ion-channel in intracellular Ca2+-release.Supported by BHF, EPSRC, Japan Society for Promotion of Science

Is the Gain of Hemichannel Activity a Common Feature Shared by Cx26 Syndromic Deafness Mutants?

Gap junctions and hemichannels (HCs) are major pathways for intercellular signaling. Both are made of homologous proteins named connexins. Mutations in the gene encoding Cx26 account for a large proportion of genetic deafness. These can lead to non-syndromic and syndromic deafness. The syndromic phenotype also includes awful skin disease. Gain of hemichannel activity has been proposed as the pathogenic mechanism behind this syndrome. Here, to test this hypothesis we used HeLa cells transiently expressing the Cx26 non-syndromic (G12V) and the syndromic (G12R, N14Y and S17F), all located in the N-termini segment. Hemichannel activity was evaluated by measure the plasma membrane permeability to fluorescent tracers, YO-PRO (MW 375.5 charge +2) and ethidium (MW 314.4; charge +1) and single channels conductances by whole cell patch clamp. Bathed in a divalent free cations solution HeLa cells expressing the syndromic G12R mutant show four and five folds increase in the uptake of YOPRO and ethidium, respectively, when compared to cells expressing wild type Cx26 (WT Cx26). At the single channel level, the full open state of the G12R mutant was around 600 pS; almost twice of the WT Cx26. On the other hand, the non-syndromic G12V and syndromic N14Y and S17F (most severe clinical phenotype) behave like the WT Cx26. These findings suggest that most Cx26 syndromic mutants in the N-termini mediate their pathogenicity by other mechanism than gain in hemichannel activity. Supported by: Millenium Institute-CINV, FONDECYT 1090573 and Anillo ACT-1104 to ADM; IEG is supported by CONICYT.

Structural Transition of N-Terminal Extension of Cardiac Troponin-I Complexed with Troponin-C caused by PKC-Mediated Phosphorylation, as Revealed by Spin Labeling EPR

Cardiac troponin (cTn) complex consisting of cTnI, cTnC and cTnT is responsible for cardiac mechanotransduction. Aberrent phosophorylation of cTnI by PKC on S42/44 or its phosphorylation mimic mutant S42/44D decreases Ca2+ sensitivity and tension force, consequently generating heart failure. Until now, consequence of cTnI by PKC phosphorylation on molecular level and associated structural mechanisms involved in it are still elusive. Combined with previously published NMR spectra and our bioinformatics analysis, this distinct N-extension of cTnI is strongly shown to be intrinsically disordered. To simulate the possible conformation of this intrinsically disordered region and potential conformational transition caused by PKC when binding with cTnC, Monte Carlo simulation was implemented to explore potential docking sites on cTnC, of which all the predicted structures were evaluated by total and binding energy with the criteria under RosettaCommons software suite. We screened around 10,000 structures for cTnC-I and cTnC-I (S42/44D) complexes, respectively. For cTnC-I complex, flexible N-extension of cTnI docked on N-domain of cTnC, putatively assisting to stabilize the full-open state of cTnC which binds strongly the regulatory region of cTnI to initiate muscle contraction. In contrast, as for cTnC-I (S42/44D), it localized on C-domain of cTnC, thus deficiently to maintain full-open state of cTnC, causing a weak interaction between the regulatory region of cTnI (S42/44D) and cTnC to generate heart failure. To verify our prediction, we are measuring residual mobility and inter-residual distance from spin labels placed on cTnI or cTnI (S42/44D) and cTnC. Preliminary mobility measurement of MSL-labeled cTnC(C84) indicated loose interaction between the regulatory region of cTnI (S42/44D) and cTnC. Measurements on the other sites are in progress.

Properties of single two‐pore domain TREK‐2 channels expressed in mammalian cells

TREK-2 (K2P10.1), a member of the two-pore domain K+ (K2P) channel family, provides the background K+ conductance in many cell types, and is a target of neurotransmitters that act on receptors coupled to Gs and Gq. We report here that TREK-2 exhibits small (TREK-2S) and large (TREK-2L) conductance phenotypes when expressed in mammalian cell lines (COS-7, HEK293, HeLa) and in Xenopus oocytes. TREK-2S phenotype shows a noisy open state with a mean conductance of 54 pS (+40 mV). TREK-2L phenotype shows a full open state (202 pS) with several short-lived sub-conductance levels. Both phenotypes were strongly activated by arachidonic acid, membrane stretch (-40 mmHg) and intracellular acidification (pH 6.4). Phosphorylation of TREK-2 produced by treatment of cells with activators of protein kinases A and C, and okadaic acid (a serine/threonine phosphatase inhibitor) decreased the current contributed by TREK-2S and TREK-2L, and caused partial switching of conductance levels from those of TREK-2S and TREK-2L to more intermediate values. Under this condition, TREK-2 exhibited six conducting levels and one closed level. TREK-2 mutants in which putative protein kinases A and C phosphorylation sites were mutated to alanines (S326A, S359A, S326A/S359A) displayed mostly TREK-2S and TREK-2L phenotypes. However, S326D and S359D mutants (as well as the double mutants) that mimic the phosphorylated state showed all six conducting levels and low channel activity. The S326A and S359A mutants did not significantly affect the intrinsic voltage dependence of TREK-2 in Mg2+-free solution. Phenotypes resembling TREK-2S and TREK-2L were also observed in cerebellar granule neurons that express TREK-2 mRNA. These results show that TREK-2 exhibits two primary modes of gating that give rise to two channel phenotypes under dephosphorylated conditions, and that its phosphorylation shifts the gating mode to include intermediate conducting levels. This represents a novel mechanism by which receptor agonists modulate the function of a K+ channel to alter cell excitability.

Multiple Conductance Substates in Pharmacologically Untreated Na+ Channels Generating Persistent Openings in Rat Entorhinal Cortex Neurons

Na(+)-channel activity recorded in cell-attached patches from entorhinal cortex neurons in the absence of gating-modifying drugs was examined to determine the possible occurrence of substate openings. Brief sojourns to subconductance levels were occasionally observed within prolonged ("persistent") burst openings. Subconductance occurrence and amplitude were determined following two distinct, complementary approaches: (1) direct visual inspection and (2) automated detection by application of a method that exploits the current variance of fixed-width tracing segments to sort amplitude estimations. The two approaches led to comparable results. At least six subconductance levels in addition to the full open state were revealed, with amplitudes that were approximately 20%, 30%, 40%, 50%, 60% and 75% that of full openings. The global probability of subconductance opening occurrence within a burst as well as the probability of observing one particular subconductance level within a burst showed no clear dependence upon membrane potential in the -40 to +10 mV range. Open- and closed-time distributions of substate openings could either be similar to those observed in burst full openings or show distinct patterns. Low-amplitude late openings were also observed in isolation, separately from full-size openings. These openings corresponded to conductance levels very similar to those of the substates observed within full-size burst openings; therefore, they were interpreted as isolated subconductance openings. Early, transient openings responsible for the fast-inactivating whole-cell Na(+)-current component also manifested distinct conductance levels, the two most prominent of which were in an approximate 75:100 amplitude ratio. Interestingly, the 75% conductance level observed among early openings occurred much more frequently than in "persistent" burst openings. We conclude that pharmacologically untreated Na(+) channels from native neurons generate substate openings that may influence differently the multiple gating modes displayed by these channels.

Molecular basis of calcium regulation in connexin-32 hemichannels.

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca2+ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca2+ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of approximately 90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels ( approximately 18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca2+ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca2+ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca2+ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.

Maurocalcine and Peptide A Stabilize Distinct Subconductance States of Ryanodine Receptor Type 1, Revealing a Proportional Gating Mechanism

Maurocalcine (MCa) isolated from Scorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III loop peptide (termed peptide A (pA)) of alpha(1s)-dihydropyridine receptor and are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for skeletal muscle EC coupling. The relationship between the actions of synthetic MCa (sMCa) and pA on RyR1 were examined. sMCa released Ca(2+) from SR vesicles (EC(50) = 17.5 nm) in a manner inhibited by micromolar ryanodine or ruthenium red. pA (0.5-40 microm) failed to induce SR Ca(2+) release. Rather, pA enhanced Ca(2+) loading into SR and fully inhibited Ca(2+)-, caffeine-, and sMCa-induced Ca(2+) release. The two peptides modified single channel gating behavior in distinct ways. With Cs(+)-carrying current, 10 nm to 1 microm sMCa induced long lived subconductances having 48% of the characteristic full open state and occasional transitions to 29% at either positive or negative holding potentials. In contrast, pA stabilized long lived channel closures with occasional burst transitions to 65% (s1) and 86% (s2) of the full conductance. The actions of pA and sMCa were observed in tandem. sMCa stabilized additional subconductance states proportional to pA-induced subconductances (i.e. 43% of pA-modified s1 and s2 substates), revealing a proportional gating mechanism. [(3)H]Ryanodine binding and surface plasmon resonance analyses indicated that the peptides did not interact by simple competition for a single class of mutually exclusive sites on RyR1 to produce proportional gating. The actions of sMCa were also observed with ryanodine-modified channels and channels deficient in immunophilin 12-kDa FK506-binding protein. These results provide evidence that sMCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance.

Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

Large tetraalkyl ammonium cations produce a reduced conductance state in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel

The purified Ca(2+)-release/ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) functions as a calcium-activated cation-selective channel under voltage clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effect of large tetraalkyl ammonium (TAA) cations, (CnH2n+1)4N+ (n = 4 and 5) on monovalent cation conduction. These cations modify the conductance of the receptor channel at positive holding potentials from the cytosolic side of the channel. Under these conditions, openings are resolved as a mixture of normal full amplitude events and events of reduced conductance. The amplitude of the reduced conductance state is a fixed proportion of the normal open state. As a proportion of all open events, the occurrence of the tetrabutyl ammonium (TBA+) related subconductance state increases with concentration and increasingly positive holding potential. The TBA+ related subconductance state displays similar conduction properties to the unmodified channel; with a linear current-voltage relationship, a similar affinity for K+ and voltage-dependent block by TEA+. A method was used to quantify the voltage dependence of the occurrence of the TBA+ effect, which yielded an effective gating charge of 1.66. A second method based on kinetic analysis of the voltage dependence of transitions between the full open state and the TBA+ related subconductance state produced a similar value. In addition, this analysis revealed that the bulk of the voltage-dependence resided in the off rate. TBA+ related subconductance events, expressed as a proportion of all open events, saturated with increasing TBA+ concentration. Kinetic analysis revealed that this could be entirely accounted for by changes in the on rate. Tetrapentyl ammonium (TPeA+) causes a qualitatively similar effect with a subconductance state of lower amplitude. The voltage-dependence of the effect was comparable to that displayed by TBA+. These findings are interpreted as a form of partial block in which more than one large TAA cation binds at the extremity of the voltage drop to produce an electrostatic barrier for ion translocation.

Characterization of large-conductance chloride channels in rabbit colonic smooth muscle.

1. A large-conductance Cl- channel was characterized in cell-free membrane patches from the rabbit longitudinal colonic smooth muscle using the patch clamp technique. In addition, the regulation of these channels by neurokinin-1 (NK-1) receptor agonists and G proteins was studied. 2. No spontaneous channel activity was observed in cell-attached patches at the cell resting potential, or in excised patches at pipette potentials (Vp) between -20 and 20 mV. In excised patches, channel activity could be induced in thirty-six out of ninety-six patches by holding the patch at Vp values more negative than -60 mV or more positive than 60 mV. Once induced, the channel showed a bell-shaped voltage activation curve in high symmetric [Cl-], with maximal open probability between 20 and -5 mV. Varying cytosolic calcium concentration ([Ca2+]) between 5 x 10(-8) M and 1.0 mM had no effect on the voltage activation of the channel. 3. In inside-out and outside-out patches, when pipette and bath solutions contained equal [Cl-] (130 mM), the anion channel showed a linear current-voltage (I-V) relationship between -60 and 60 mV with a slope conductance of 309 +/- 20 pS (n = 13). Reversal potential measurements indicated that the channel was selective for Cl- over Na+ and K+ (PCl/PNa = 6:1). 4. Channel openings from the closed state to the full open state as well as transitions through smaller conductance states were observed. The smallest detectable substate had a conductance of 15.6 pS. Based on the similarities in selectivity and linearity of the I-V curve of the smaller conductances with the full open state, and kinetic analysis of channel activity, it is concluded that the large conductance channel is composed of multiple substates which can either open and close independently, or simultaneously via a main gate. 5. The stilbene derivative diiso-thiocyanato-stilbene-disulphonic acid (DIDS) and the diphenylamine-2-carboxylate analogue 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused a dose-dependent, reversible flicker block of the small conductance and significantly reduced the macroscopic current flow through the channel. 6. In quiescent outside-out patches, when the pipette contained a 140 mM-CsCl solution with 10(-6) M-CaCl2, 1.2 mM-MgCl2 and 1 mM-GTP, and the bath contained Ringer solution, addition of the NK-1 receptor antagonists substance P methylester resulted in activation of the full conductance state and of smaller substates.(ABSTRACT TRUNCATED AT 400 WORDS)