Macroscopic Conductance Research Articles

Fungicidal Activities and Mechanisms of Action of Pseudomonas syringae pv. syringae Lipodepsipeptide Syringopeptins 22A and 25A

The plant-associated bacterium Pseudomonas syringae pv. syringae simultaneously produces two classes of metabolites: the small cyclic lipodepsinonapeptides such as the syringomycins and the larger cyclic lipodepsipeptide syringopeptins SP22 or SP25. The syringomycins inhibit a broad spectrum of fungi (but particularly yeasts) by lipid-dependent membrane interaction. The syringopeptins are phytotoxic and inhibitory to Gram-positive bacteria. In this study, the fungicidal activities of two major syringopeptins, SP22A and SP25A, and their mechanisms of action were investigated and compared to those of syringomycin E. SP22A and SP25A were observed to inhibit the fungal yeasts Saccharomyces cerevisiae and Candida albicans although less effectively than syringomycin E. S. cerevisiae mutants defective in ergosterol and sphingolipid biosyntheses were less susceptible to SP22A and SP25A but the relative inhibitory capabilities of SRE vs. SP22A and SP25A were maintained. Similar differences were observed for capabilities to cause cellular K+ and Ca2+ fluxes in S. cerevisiae. Interestingly, in phospholipid bilayers the syringopeptins are found to induce larger macroscopic ionic conductances than syringomycin E but form single channels with similar properties. These findings suggest that the syringopeptins target the yeast plasma membrane, and, like syringomycin E, employ a lipid-dependent channel-forming mechanism of action. The differing degrees of growth inhibition by these lipodepsipeptides may be explained by differences in their hydrophobicities. The more hydrophobic SP22A and SP25A might interact more strongly with the yeast cell wall that would create a selective barrier for their incorporation into the plasma membrane.

Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate

The CFTR contributes to Cl⁻ and HCO₃⁻ transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl⁻ and HCO₃⁻ in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl⁻ and HCO₃⁻ regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO₃⁻ than when it contains Cl⁻. This difference appears to reflect differences in the ability of extracellular HCO₃⁻ and Cl⁻ to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO₃⁻ concentrations and membrane potentials and can result in up to ∼50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.

The Role of Amino Terminus of Mouse Cx50 in Determining Transjunctional Voltage-Dependent Gating and Unitary Conductance

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating ( V j-gating) and the unitary gap junction channel conductance ( γ j). To better understand the molecular structure(s) determining the V j-gating properties and the γ j of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to V j ( G j/ V j relationship), slowed V j-gating kinetics, and reduced γ j as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar V j-gating properties and γ j to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The G j/ V j relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance ( G min) was near zero. Moreover, the single Cx50N9R channel only displayed one open state ( γ j = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the V j-gating and the γ j, and the introduction of a positively charged Arg at the ninth position reduced the G min with a correlated disappearance of the substate at the single channel level.

Substrate-induced strain and its effect in CrO2 thin films

We report a study of substrate-induced strain and its effect in (100) and (110) CrO2 thin films deposited on TiO2 substrates of respective orientations. While the (110) CrO2 films grow essentially strain-free, the (100) CrO2 films were found to be strained in all lattice directions—out of plane direction was compressively strained while in-plane directions were under tensile strain. Crystal lattice parameters were determined in strained (100) and strain-free (110) CrO2 films together with the amount of strain in the three lattice directions. We found substrate-induced strain to significantly affect the magnetic moment in the (100) CrO2 films at room temperature—reducing the magnetic moment with increasing strain in the (100) films while strain-free (110) CrO2 thin films have higher moments for all thicknesses. Qualitative macroscopic conductance behavior in the strained (100) and strain-free (110) CrO2 films were found to be comparable for temperatures in the range of 5–400 K, showing similar behavior at low temperature as well as near Tc.

Inhibition of the activation pathway of the T-type calcium channel Ca V3.1 by ProTxII

Toxins have been used extensively to probe the gating mechanisms of voltage-gated ion channels. Relatively few such tools are available to study the low-voltage activated T-type Ca channels, which underlie thalamic neuron firing and affect sleep, resistance to seizures, and weight gain. Here we show that ProTxII, a peptide toxin recently isolated from the venom of the tarantula spider Thrixopelma pruriens, dose-dependently inhibited Ca V3.1 causing a decrease in current (81.6% ± 3.1% at −30 mV in 5 μM toxin) and a positive shift in the voltage range of activation (+34.5 mV ± 4.4 mV). Toxin-modified currents were slower to activate and faster to deactivate and they displayed a longer lag in the onset of current, i.e. the Cole-Moore shift, consistent with the inhibition of gating transitions along the activation pathway, particularly the final opening transition. Single-channel current amplitude and total gating charge were unaffected by toxin, ruling out a change in ion flux or channel dropout as mechanisms for the decrease in macroscopic conductance. A positive shift in the voltage range of gating charge movement (+30.6 mV ± 2.6 mV shift in the voltage of half maximal charge movement in the presence of 5 μM toxin) confirmed that ProTxII-induced gating perturbations in this channel occur at the level of the voltage sensors, and kinetic modeling based on these findings suggested that reductions in current magnitude could be largely accounted for by kinetic perturbations of activation.

A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity

ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased the macroscopic conductance ratio, GRb/GK by 17 ± 3 fold and altered the selectivity sequence from NH4 > K > Tl > Rb >> Cs in wt-Kir1.1 to: Rb > Cs > Tl > NH4 >> K in R128Y, without significant change in the high K/Na permeability ratio of Kir1.1. R128M produced similar, but smaller, increases in Rb, Tl, NH4 and Cs conductance relative to K. R128Y remained susceptible to block by both external Ba and the honeybee toxin, TPNQ, although R128Y had a reduced affinity for TPNQ, relative to wild-type. The effect of R128Y-Kir1.1b on the GRb/GK ratio can be partly explained by a larger single-channel Rb conductance (12.4 ± 0.5 pS) than K conductance (< 1.5 pS ) in this mutant. The kinetics of R128Y gating at -120 mV with Rb as the permeant ion were similar to those of wt-Kir1.1 conducting Rb, but with a longer open time (129 ms vs. 6 ms for wt) and two closed states (13 ms, 905 ms), resulting in an open probability (Po) of 0.5, compared to a Po of 0.9 for wt-Kir1.1, which had a single closed state of 1 ms at -120mV. Single-channel R128Y rectification was eliminated in excised, inside-out patches by removal of internal Mg and polyamines. The large increase in the Rb/K conductance ratio, with no change in K/Na permeability or rectification, is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter.

Isoform‐dependent modification of T‐type calcium channels by ProTxII

The 3 isoforms of the T‐type calcium (Ca) channel are differentially expressed in the nervous system and within individual neurons (Perez‐Reyes 2003; McKay et al. 2006). There exist few experimental tools that can differentiate between channel isoforms. Here, we show that ProTxII, a highly charged peptide toxin from the venom of the tarantula Thrixopelma pruriens, differentially modifies the 3 isoforms. CaV3.1 and CaV3.3 channels are sensitive to the toxin when expressed heterologously in HEK cells. In CaV3.1, 2μM toxin produces a positive, dose‐dependent shift in the voltage range of activation of ~20mV and a reduction in macroscopic conductance of ~50%. In CaV3.3 channels 2.5μM ProTxII minimally shifted activation by ~4mV but reduced conductance by ~50%. In CaV3.2, 5μM toxin produces a minimal shift of ~5mV but no reduction in conductance. We examined the sequences of CaV3.1 and CaV3.2 for charge differences in extracellular linkers and p‐loops which may contribute to differential isoform sensitivity. We examined three reciprocal pairs in DIIS3, DII p‐loop, and DIIIS3 of these channels. Reciprocal mutations in the domain II p‐loop profoundly affected toxin sensitivity. In the CaV3.1 background the double mutation E894K/R895T resulted in reduced sensitivity to ProTxII. Conversely the K944E/T945R mutation in the CaV3.2 background conferred sensitivity. Supported by APS Summer Research Fellowship (EOH).

Thermal contact conductance characterization via computational contact homogenization: A finite deformation theory framework

AbstractIn order to predict the macroscopic thermal response of contact interfaces between rough surface topographies, a computational contact homogenization technique is developed at the finite deformation regime. The overall homogenization framework transfers macroscopic contact variables, such as surfacial stretch, pressure and heat flux, as boundary conditions on a test sample within a micromechanical interface testing procedure. An analysis of the thermal dissipation within the test sample reveals a thermodynamically consistent identification for the macroscopic thermal contact conductance parameter that enables the solution of a homogenized thermomechanical contact boundary value problem based on standard computational approaches. The homogenized contact response effectively predicts a temperature jump across the macroscale contact interface. The strong dependence of this homogenized response on macroscale solution variables of interest is demonstrated via representative three‐dimensional numerical investigations. The proposed contact homogenization framework is suitable for the analysis of similar energy transport phenomena across heterogeneous contact interfaces where the investigation of the sources for energy dissipation is of concern. Copyright © 2010 John Wiley &amp; Sons, Ltd.

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

Micronutrients such as siderophore-bound iron and vitamin B(12) cross the outer membrane of gram-negative bacteria through a group of 22-stranded beta-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the beta-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.

Improving cardiac gap junction communication as a new antiarrhythmic mechanism: the action of antiarrhythmic peptides

Co-ordinated electrical activation of the heart is maintained by intercellular coupling of cardiomyocytes via gap junctional channels located in the intercalated disks. These channels consist of two hexameric hemichannels, docked to each other, provided by either of the adjacent cells. Thus, a complete gap junction channel is made from 12 protein subunits, the connexins. While 21 isoforms of connexins are presently known, cardiomyocytes typically are coupled by Cx43 (most abundant), Cx40 or Cx45. Some years ago, antiarrhythmic peptides were discovered and synthesised, which were shown to increase macroscopic gap junction conductance (electrical coupling) and enhance dye transfer (metabolic coupling). The lead substance of these peptides is AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)), a peptide with a horseshoe-like spatial structure as became evident from two-dimensional nuclear magnetic resonance studies. A stable D: -amino-acid derivative of AAP10, rotigaptide, as well as a non-peptide analogue, gap-134, has been developed in recent years. Antiarrhythmic peptides act on Cx43 and Cx45 gap junctions but not on Cx40 channels. AAP10 has been shown to enhance intercellular communication in rat, rabbit and human cardiomyocytes. Antiarrhythmic peptides are effective against ventricular tachyarrhythmias, such as late ischaemic (type IB) ventricular fibrillation, CaCl(2) or aconitine-induced arrhythmia. Interestingly, the effect of antiarrhythmic peptides is higher in partially uncoupled cells and was shown to be related to maintained Cx43 phosphorylation, while arrhythmogenic conditions like ischaemia result in Cx43 dephosphorylation and intercellular decoupling. It is still a matter of debate whether these drugs also act against atrial fibrillation. The present review outlines the development of this group of peptides and derivatives, their mode of action and molecular mechanisms, and discusses their possible therapeutic potential.

Non-Newtonian Fluid Flow through Three-Dimensional Disordered Porous Media

We investigate the flow of various non-newtonian fluids through three-dimensional disordered porous media by direct numerical simulation of momentum transport and continuity equations. Remarkably, our results for power-law (PL) fluids indicate that the flow, when quantified in terms of a properly modified permeability-like index and Reynolds number, can be successfully described by a single (universal) curve over a broad range of Reynolds conditions and power-law exponents. We also study the flow behavior of Bingham fluids described in terms of the Herschel-Bulkley model. In this case, our simulations reveal that the interplay of (i) the disordered geometry of the pore space, (ii) the fluid rheological properties, and (iii) the inertial effects on the flow is responsible for a substantial enhancement of the macroscopic hydraulic conductance of the system at intermediate Reynolds conditions.

Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A's impact in a firing-neuron model, suggest that syntaxin 1A's interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation.

Phosphorylation of connexin43 on serine 306 regulates electrical coupling

Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia.

Auto-phosphorylation of a voltage-gated K+ channel controls non-associative learning

Here, we characterize a new K(+) channel-kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore-forming subunit, dubbed KHT-1 (73% homology to human Kv3.1), and the accessory subunit MPS-1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT-1 and MPS-1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT-1 is a substrate for the kinase activity of MPS-1. Behavioural analysis further shows that the kinase activity of MPS-1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS-1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT-1-MPS-1 seems to be linked to distinct behavioural responses. In the non-phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto-phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.

Unitary Recordings of TRP and TRPL Channels From Isolated Drosophila Retinal Photoreceptor Rhabdomeres: Activation by Light and Lipids

Transient receptor potential (TRP) channels play key roles in sensory transduction. The TRP family founding members, the Drosophila light-dependent channels, were previously studied under voltage clamp, but had not been characterized in intact rhabdomeres at single-channel level. We report patch-clamp recordings from intact isolated photoreceptors of wt and mutant flies lacking TRP (trp(343)), TRPL (trpl(302)), or both channels (trp(313); trpl(302)). Unitary currents were activated by light in rhabdomere-attached patches. In excised rhabdomeral patches, the channels were directly activated by molecules implicated in phototransduction, such as diacylglycerol and polyunsaturated fatty acids. Currents recorded from trpl photoreceptors are blocked by external Ca(2+), Mg(2+) (1 mM), and La(3+) (20 muM), whereas those from trp photoreceptors are not. Rhabdomeric patches lacked voltage-dependent activity. Patches from trp;trpl mutants were devoid of channels. These characteristics match the macroscopic conductances, suggesting that the unitary currents from Drosophila trpl and trp photoreceptors correspond to TRP and TRPL.

Reconstitution Of Tonb-dependent Transporters In Planar Lipid Bilayers

Micronutrients such as siderophore-bound iron and vitamin B12 cross the outer membrane of gram-negative bacteria through a group of 22-stranded β-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the β-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel to open a space wide enough for siderophores to diffuse through.Attempts to reconstitute these plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. Recently, however, we have discovered that if the cis solution contains 4 M urea, then macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir and BtuB, and in the case of the former two, the channels were closed by removing the 4 M urea. The basic native structure of these proteins appears to be preserved by our reconstitution procedure. With FhuA, for example, addition of ferrichrome (its siderophore) to the trans side reversibly inhibits 4 M urea-induced channel opening and blocks the channels with a Kd of ∼0.5 nM, the same as that found biologically. In the case of Cir, as another example, addition of colicin Ia (the microbial toxin that targets Cir) to the trans side prevents 4 M urea-induced channel opening. Our working hypothesis is that the 4 M urea reversibly denatures the plug, causing it to either rearrange itself or to come completely out of the barrel, thereby providing a conduction pathway. Whether the 4 M urea action on the plug mimics to some extent that of TonB remains for future investigation.

Human cardiac gap-junction coupling: effects of antiarrhythmic peptide AAP10

Ventricular arrhythmia is one of the most important causes of death in industrialized countries and often accompanies myocardial infarction and heart failure. In recent years modification of gap-junctional coupling has been proposed as a new antiarrhythmic principle. We wanted to examine whether the gap junction modulator (antiarrhythmic peptide) AAP10 exerts effects on human cardiac gap junctions, whether the effect might be enhanced in uncoupled cells, whether it affects electrical and metabolic coupling, and which of the cardiac connexin isoforms (Cx40, Cx43, Cx45) may be affected. We determined the influence of 50 nM AAP10 (H(2)N-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH(2)) on macroscopic gap junction conductance by dual whole-cell voltage clamping in human and rat cardiomyocytes. Cells were partially uncoupled by CO(2)-mediated acidosis (pH 6.3) or kept at 'normal' conditions (pH 7.4, T 36 degrees C). Furthermore, we investigated effects of AAP10 in HeLa cells stably transfected with connexin 40, 43, or 45 and on metabolic coupling determined by dye transfer (Lucifer yellow). AAP10 (50 nM)-enhanced gap-junctional intercellular coupling in human and rat cardiomyocytes, completely prevented CO(2)-acidosis-induced uncoupling and improved metabolic coupling. The coupling effect of AAP10 was significantly enhanced in previously uncoupled cells. Regarding the connexin isoforms, AAP10-enhanced electrical and metabolic coupling in HeLa cells expressing Cx43 or Cx45, but not in HeLa cells expressing Cx40. We conclude that the antiarrhythmic peptide AAP10, which improves gap-junctional intercellular coupling and prevents uncoupling by acidification in human cardiomyocytes, might be useful for antiarrhythmic strategies regarding arrhythmias caused by uncoupling of Cx43 and Cx45, but not Cx40.

External Ba 2+ Block of Human Kv1.5 at Neutral and Acidic pH: Evidence for H o+-Induced Constriction of the Outer Pore Mouth at Rest

Previous studies have shown that low pH o accelerates depolarization-induced inactivation and decreases the macroscopic conductance by reducing channel availability. To test the hypothesis that outer pore constriction underlies the decreased conductance at low pH o, external Ba 2+ was used to examine the accessibility of the channel pore at rest under neutral and acidic conditions. At pH o 7.4, Ba 2+ block of closed channels follows a monoexponential time course and involves a low-affinity superficial site ( K D ≅ 1 mM, −80 mV, 0 mM K o + ) and a high-affinity site ( K D ≅ 4 μM) deeper in the pore. Depolarization promotes Ba 2+ dissociation and an analytical model incorporating state-dependent changes of Ba 2+ affinity is presented that replicates the frequency dependence of the time course and the extent of block. Open-channel block by Ba 2+ is weak. At pH o 5.5, both the access to and exit from the deep site is inhibited. These results are consistent with a low-pH o-induced conformational change in the outer pore that prevents Ba 2+ binding at rest or unbinding during depolarization. If a pore constriction is involved, similar to that proposed to occur during P/C-type inactivation, this would imply that closed-state inactivation in Kv1.5 occurs under acidic conditions.

Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release

Systemic osmoregulation is an integrated physiological process through which water intake and excretion are continuously balanced against salt intake and excretion to maintain the osmolality of the extracellular fluid near an optimal 'set-point' value. The behaviors (that is, thirst and sodium appetite) and renal responses (diuresis and natriuresis) that are modulated to mediate osmoregulatory homeostasis are mainly controlled by the nervous system. Appropriate regulation of these parameters depends in large part on specialized osmosensitive neurons, termed osmoreceptors, which convert changes in plasma osmolality into electrical signals that ultimately modulate effector functions to achieve homeostasis. Previous work has shown that mechanosensitive cation channels expressed in osmoreceptor neurons play a key role in the process of osmosensory transduction. Although the molecular identity of these channels remains unknown, a growing body of evidence, reviewed here, indicates that members of the transient receptor potential vanilloid family of ion channels may contribute to osmosensory transduction and to homeostatic responses implicated in the control of water balance.

A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.