Open Time Constants Research Articles

Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation.

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl- channel.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP. To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH. We found that short-lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP-binding site 1 formed by the nucleotide-binding domains. Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl- channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP-binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single-channel activity of human CFTR at different intracellular pH (pHi ) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open-time constant (τo ) of wild-type CFTR. By contrast, the fast closed-time constant (τcf ), which describes the short-lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three-state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo , τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP-binding site 1, including K464A, D572N and the CF-associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP-dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP-binding site 1 modulates intraburst gating.

Robust controller design for automated kick handling in managed pressure drilling

The problem considered in this paper is that of controlling downhole pressure during oil and gas well drilling, with a particular focus on handling gas kicks leading to two-phase gas–liquid flow conditions. We identify a first-order approximation to the infinite-dimensional system which captures the dominating mode of the pressure dynamics in the frequency range of interest, while the high-frequency pressure dynamics are represented by a multiplicative uncertainty. This approximation is then modified to accommodate the changes to the dynamics introduced by the two-phase flow. The linearized plant has an open-loop time constant which varies between 2 and 600s depending on operating point and gas distribution in the well. Robust controller design is then performed using linear matrix inequalities (LMIs) via a polytopic norm-bounded description of both the high-frequency multiplicative, and the low-frequency parametric uncertainty. It is shown that, in order to achieve acceptable performance over such a large range of open loop time constants, a time-varying controller gain is required. The main contribution of the paper is to achieve this control objective systematically by formulating the control design problem as an LMI optimization problem. Then optimal solutions of the LMI problem can be obtained in polynomial time by using modern interior point method (IPM) numerical solution algorithms.

Role of protein phosphatases in the run down of guinea pig cardiac Cav1.2 Ca2+ channels.

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca(2+) channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca(2+) channel currents with the patch-clamp technique. Calmodulin (CaM) and ATP were used to restore channel activity in inside-out patches. Inhibitors of protein phosphatases were applied to investigate the role of phosphatases. The specific protein phosphatase type 1 (PP1) inhibitor (PP1 inhibitor-2) and protein phosphatase type 2A (PP2A) inhibitor (fostriecin) abolished the slow run down of Cav1.2 Ca(2+) channels, which was evident as the time-dependent attenuation of the reversing effect of CaM/ATP on the run down. However, protein phosphatase type 2B (PP2B, calcineurin) inhibitor cyclosporine A together with cyclophilin A had no effect on the channel run down. Furthermore, PP1 inhibitor-2 mainly prolonged the open time constants of the channel, specifically, the slow open time. Fostriecin primarily shortened the slow close time constants. Our data suggest that PP1 and PP2A were involved in the slow phase of Cav1.2 Ca(2+) channel run down. In addition, they exerted different effects on the open-close kinetics of the channel. All above support the view that PP1 and PP2A may dephosphorylate distinct phosphorylation sites on the Cav1.2 Ca(2+) channel.

An Online Simplified Nonlinear Controller for Transient Stabilization Enhancement of DFIG in Multi-Machine Power Systems

An adaptive nonlinear controller for transient stability and voltage regulation of power systems based DFIG in multimachine configuration is presented using a standard third order dynamical model of the DFIG. Finite time estimators for the unmeasurable time derivative of the quadrature component of the DFIG stator current, mechanical input, unknown direct axis transient open circuit time constant (function of the rotor resistance) are presented. The main feature of the proposed control scheme is its robustness with respect to large perturbations and parameter variations. Numerical results are presented to illustrate the performance of the proposed control scheme and its robustness properties.

Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.

Pregnenolone sulfate (PS), one of the most commonly occurring neurosteroids in the central nervous system, influences the function of several receptors. PS modulates N-methyl-D-aspartate receptors (NMDARs) and has been shown to have both positive and negative modulatory effects on NMDAR currents generally in a subtype-selective manner. We assessed the gating mechanism of PS modulation of GluN1/GluN2A receptors transiently expressed in human embryonic kidney 293 cells using whole-cell and single-channel electrophysiology. Only a modest effect on the whole-cell responses was observed by PS in dialyzed (nonperforated) whole-cell recordings. Interestingly, in perforated conditions, PS was found to increase the whole-cell currents in the absence of nominal extracellular Ca(2+), whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca(2+). The Ca(2+)-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca(2+)-dependent bidirectional effect of PS. Single-channel cell-attached analysis demonstrated that PS primarily affected the mean open time to produce its effects: positive modulation mediated by an increase in duration of open time constants, and negative modulation mediated by a reduction in the time spent in a long-lived open state of the receptor. Further kinetic modeling of the single-channel data suggested that the positive and negative modulatory effects are mediated by different gating steps which may represent GluN2 and GluN1 subunit-selective conformational changes, respectively. Our studies provide a unique mechanism of modulation of NMDARs by an endogenous neurosteroid, which has implications for identifying state-dependent molecules.

Characteristics of single large-conductance Ca2+-activated K+ channels and their regulation of action potentials and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus

Large-conductance Ca2(+)-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional roles of BK channels in parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal 7-9 days. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca(2+)-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS and a 50% opening probability at +27.9 mV, the channel open time constant was 3.37 ms at +20 mV, and dwell time increased exponentially as the membrane potential depolarized. At the +20-mV holding potential, the [Ca2+]50 was 15.2 μM with a P0.5 of 0.4. Occasionally, some BK channels showed a transient channel opening and fast inactivation. Using whole cell voltage clamp, we found that BK channel mediated outward currents and afterhyperpolarization currents (IAHP). Using whole cell current clamp, we found that application of BK channel blocker iberiotoxin (IBTX) increased spike half-width and suppressed fast afterhyperpolarization (fAHP) amplitude following single APs. In addition, IBTX application increased spike half-width and reduced the spike frequency-dependent AP broadening in trains and spike frequency adaption (SFA). Furthermore, BK channel blockade decreased spike frequency. Collectively, these results demonstrate that PCMNs have BK channels that significantly regulate AP repolarization, fAHP, SFA, and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.

Colonic inflammation alters Src kinase-dependent gating properties of single Ca2+ channels via tyrosine nitration

Nitration of L-type calcium channels during colonic inflammation impairs phosphorylation by the tyrosine kinase, Src kinase. This results in decreased calcium currents. The purpose of this study was to determine the mechanism of the downregulation of Ca2+ currents in colonic inflammation. In whole cell voltage clamp of mouse single smooth muscle cells, long-duration depolarization produced noninactivating calcium currents that were significantly reduced by the Src kinase inhibitor, protein phosphatase 2 (PP2). Unitary Ba2+ currents were recorded upon repolarization from positive potentials in cell-attached patches of smooth muscle and hCa(v)1.2b-transfected cells to assess the properties of the single channels attributed to the noninactivating open state. Repolarization to -40 mV from 0 mV resulted in single-channel events with conductance of approximately 23 pS. The ensemble average of the tail currents from 1,000 sweeps was 337 +/- 27 fA in control and 218 +/- 49 fA (P < 0.05) in inflamed cells. Neither open-probability nor open-time constants were significantly different between control and inflamed cells. However, the transition to the open state measured as channel availability was significantly reduced from 19 +/- 3% to 6.4 +/- 1%. Similarly, peak ensemble average current and channel availability were significantly reduced by PP2 and treatment with peroxynitrite in control cells. Mutation of COOH-terminal tyrosine residues in hCa(v)1.2b Chinese hamster ovarian cells also decreased peak ensemble average tail currents and availability. The present findings suggest that the transition of Ca2+ channels to the noninactivating open state is Src kinase dependent. Tyrosine nitration prevents Src-mediated transitions, leading to decreased calcium currents.

Src kinase‐dependent gating properties of single Ca2+ channels are altered by tyrosine nitration in colitis

Reduced L‐type Ca2+ ‐channel (LTCC) function contributes to colonic dysmotility during inflammation. We have previously shown that nitration of tyrosine residues within the carboxy terminus of LTCC (Y1837 and Y2134) impairs c‐Src kinase regulation during colitis. We examined the gating properties of single LTCC, using cell‐attached patch clamp mode, in mouse colon smooth muscle cells (control vs colitis‐TNBS) and CHO cells transfected with hCaV1.2b (wild type) or mutated hCaV1.2b (Y1837F:Y2134F). Inflammation significantly reduced LTCC ensemble average current (−0.33 vs −0.22 pA, P=0.05), without affecting the single‐channel properties: mean amplitude (−2.4pA), slope conductance (23 vs 27pS) or the open‐time constants (τ1: 1.6 vs 1.2; τ2: 6.1 vs 3.9 ms). However, the availability (slow gating) of LTCC was significantly reduced by inflammation (0.19 vs 0.06; P=0.02) without affecting the open probability of modal gating. Similar effects were seen with inhibition of c‐Src kinase by PP2 or by tyrosine nitration induced by SIN‐1+ONOO− combination in smooth muscle cells. Mutation of tyrosine residues (Y1837/2134F) in hCaV1.2b, which prevents c‐Src kinase regulation, resulted in diminished channel availability. This study demonstrates that src kinase‐ phosphorylation enhances the availability of LTCC, that is impaired by tyrosine nitration during colonic inflammation. Supported by NIH DK046367.

Beta-receptor activation increases sodium current in guinea pig heart

To study the influence of beta-receptor activation on sodium channel current and the physiological significance of increased sodium current with regard to the increased cardiac output caused by sympathetic excitation. Multiple experimental approaches, including ECG, action potential recording with conventional microelectrodes, whole-cell current measurements, single-channel recordings, and pumping-force measurements, were applied to guinea pig hearts and isolated ventricular myocytes. Isoprenaline was found to dose-dependently shorten QRS waves, increase the amplitude and the V(max) of action potentials, augment the fast sodium current, and increase the occurrence frequencies and open time constants of the long-open and burst modes of the sodium channel. Increased levels of membrane-permeable cAMP have similar effects. In the presence of a calcium channel blocker, TTX reversed the increased pumping force produced by isoprenaline. Beta-adrenergic modulation increases the inward sodium current and accelerates the conduction velocity within the ventricles by changing the sodium channel modes, which might both be conducive to the synchronous contraction of the heart and enhance its pumping function.

Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.

Single channel study of deltamethrin interactions with wild-type and mutated rat Na V1.2 sodium channels expressed in Xenopus oocytes

Single channel analysis was used to compare the electrophysiological properties of wild-type (WT) and I874M mutant (M874) rat Na V1.2 channels expressed in Xenopus oocytes and their modulation by the pyrethroid deltamethrin. In the absence of pyrethroid, histograms of channel open times were best-fit by single exponentials. The open time constants at −40 mV for WT (0.53 ± 0.05 ms) and M874 (0.65 ± 0.08 ms) channels were significantly different and both decreased with depolarisation. At ≥100 nM deltamethrin, WT open time histograms at −40 mV were best-fit by two exponentials (time constants, 0.49 ± 0.03 ms ( τ o,fast,WT) and 5.2 ± 0.5 ms ( τ o,slow,WT). The population of long-duration openings and τ o,slow,WT increased when the concentration of deltamethrin was raised, but τ o,fast,WT was unaffected. Qualitatively similar results were obtained for the M874 channel, but with ≥10 nM deltamethrin. Deltamethrin also caused a negative shift in the relationships between channel opening probability ( P op) and membrane potential and first latency and membrane potential, suggesting that the pyrethroid binds to the closed channel. Selectivity for Na was increased by the pyrethroid (10 μM, WT; 1 μM, mutant).

Iron (II) Modulation of the Cardiac Ryanodine Receptor (RyR2)

Cardiomyopathies and arrhythmias are major causes of mortality in chronic iron overload. There is evidence that iron overload impairs cardiomyocytes Ca2+ homeostasis (Baptista-Hon et al, 2005). However its molecular substrates remain unknown. Cardiac ryanodine receptors (RyR2) dysfunction is implicated in several diseases where Ca2+ homeostasis is lost. We therefore wanted to investigate RyR2 role as a potential target for iron-induced cardiomyopathies.We isolated heavy sarcoplasmic reticulum (HSR) vesicles containing RyR2 from sheep hearts. RyR2 were reconstituted into L-α-phosphatidylethanolamine (PE) bilayers. Unitary currents were measured under voltage-clamp with 250mM Cs+ as the charge carrier and 10μM activating Ca2+. High affinity [3H]ryanodine binding of the native vesicles was detected by liquid scintillation counting. Non-specific binding determined by incubations with 100x cold ryanodine.Fe2+ reduced RyR2 open probability and conductance in a dose dependent manner. Lifetime analysis revealed 5 shut times components and 3 open times components in control. Fe2+ caused an extra-shut component. Furthermore, there was a dose dependent shift in the open time constants towards the faster components. The binding assays revealed a [Fe2+] dependent, co-operative reduction in [3H]ryanodine binding to HSR vesicles. Preliminary data of [3H]ryanodine binding in increasing [Ca2+] showed a rightward shift in the presence of Fe2+.The results presented here show for the first time that Fe2+ is a potent inhibitor of RyR2. The mechanism of this inhibition may be due to competition with Ca2+ for RyR2 activation sites. Suppression of RyR2 activity by Fe2+ may therefore be one of the mechanisms involved in iron-induced cardiomyopathies.ReferencesBaptista-Hon, D, Diaz, M. E., and Elliot, A. C. Acute exposure to iron (II) alters calcium handling in isolated rat ventricular myocytes. Journal of Molecular and Cellular Cardiology 39, 179. 2005.

Two Distinct Gating Cycles of CFTR Chloride Channels

CFTR channels, at maximally effective [ATP], switch between open and closed states 1-2 s−1. Pyrophosphate (PPi), applied with ATP, slows down the gating cycle by locking the channel in a stable open state (τo ∼30 s). PPi alone, applied immediately following closing of ATP-opened channels, locks open the channel with the same open time constant. However, the open state induced by PPi long after ATP removal (> 2 min) assumes a lifetime of 1.5 s, indicating the presence of two different closed states with distinct responses to PPi. By altering the duration of ATP removal and measuring the response of closed channels to PPi, we estimated the lifetime of the closed state (C∗) that enters the lock-open state to be ∼30 s. Since the lifetime of the C∗ state can be modulated by N6-phenylethyl-ATP (P-ATP), a high-affinity ATP analog, or by mutations that lower the ATP binding affinity at NBD1, we propose that one ATP molecule remains tightly bound at NBD1 during this closed state. As the trapped ATP molecule should survive for numerous gating cycles before being replaced by a second ligand, we carried out single channel experiments where the perfusion solution containing 2 mM ATP (ropen = 3.33 ± 0.19 s−1; τopen = 235 ± 19.8 ms) was directly (dead time ∼40 ms) switched to one with 50 μM P-ATP. The Po of the channel increases in two steps. The channel opening rate was immediately increased (ropen = 4.88 ± 0.45 s−1) upon solution exchange, while the open time was not prolonged until ∼40s after the application of P-ATP (τopen = 386.7 ± 25.2 ms). This result indicates two gating cycles; one is sorely driven by fast ATP binding/hydrolysis in NBD2 while another involves slow dissociation of ATP in NBD1.

Elementary properties of Kir2.1, a strong inwardly rectifying K + channel expressed by pigeon vestibular type II hair cells

By using the patch-clamp technique in the cell-attached configuration, we have investigated the single-channel properties of an inward rectifier potassium channel (Kir) expressed by pigeon vestibular type II hair cells in situ. In high-K + external solution with 2 mM Mg 2+, Kir inward current showed openings to at least four amplitude levels. The two most frequent open states (L2 and L3) had a mean slope conductance of 13 and 28 pS, respectively. L1 (7 pS) and L4 (36 pS) together accounted for less than 6% of the conductive state. Closed time distributions were fitted well using four exponential functions, of which the slowest time constant (τ C4) was clearly voltage-dependent. Open time distributions were fitted well with two or three exponential functions depending on voltage. The mean open probability (P O) decreased with hyperpolarization (0.13 at −50 mV and 0.03 at −120 mV). During pulse-voltage protocols, the Kir current-decay process (inactivation) accelerated and increased in extent with hyperpolarization. This phenomenon was associated with a progressive increase of the relative importance of τ C4. Kir inactivation almost disappeared when Mg 2+ was omitted from the pipette solution. At the same time, P O increased at all membrane voltages and the relative importance of L4 increased to a mean value of 47%. The relative importance of τ C4 decreased for all open states, while L4 only showed a significantly longer open time constant. The present work provides the first detailed quantitative description of the elementary properties of the Kir inward rectifier in pigeon vestibular type II hair cells and specifically describes the Kir gating properties and the molecule's sensitivity to extracellular Mg 2+ for all subconductance levels. The present results are consistent with the Kir2.1 protein sustaining a strong inwardly rectifying K + current in native hair cells, characterized by rapid activation time course and slow partial inactivation. The longest closed state (τ C4) appears as the main parameter involved in time- and Mg 2+-dependent decay. Finally, in contrast to Kir2.1 results described so far for mammalian cells, external Mg 2+ had no effect on channel conductance.

The Two ATP Binding Sites of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Play Distinct Roles in Gating Kinetics and Energetics

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase–dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

Properties of a potassium channel in the basolateral membrane of renal proximal convoluted tubule and the effect of cyclosporine on it

We studied the potassium channel in the basolateral membrane of the rat proximal convoluted tubule as affected by cyclosporine A. Proximal convoluted tubules were dissected from the rat kidney under a stereoscopic microscope, without a preliminary enzyme treatment. The standard configuration for single-channel tight seal patch-clamp technique was used to record channel currents. A small conductance, stretch-sensitive potassium channel could be observed spontaneously in most of the cell-attached patches as the gigaohm seal was formed. In the inside-out configuration, channel activity was diminished. The K(+) channel appeared to be an inward rectifier. The limiting inward slope conductance was 28.3+/-1.7 pS (Vp was between 40 mV and 80 mV, n=6) and the outward chord conductance was 5.6+/-0.3 pS (Vp was between -40 and -60 mV, n=5). The open dwell time constants of the potassium channel were 0.524 ms and 5.087 ms, while the closed dwell time constants were 1.029 ms and 16.500 ms. The opening probability of the channel decreased when the extracellular fluid was acidified. Cyclosporine A had no significant effect on the potassium channel of the proximal tubular cell in the basolateral membrane at concentrations of 10 and 50 microg/ml, while at 100 microg/ml, it decreased the opening probability.

Cav1.4 Encodes a Calcium Channel with Low Open Probability and Unitary Conductance

When transiently expressed in tsA-201 cells, Cav1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100mM external barium as the charge carrier to determine the single-channel properties of Cav1.4 and to compare them to those of the Cav1.2. Cav1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Cav1.4 and Cav1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of −20 to +20mV. The single-channel conductance of Cav1.4 channels was ∼4pS compared with ∼20pS for Cav1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Cav1.4 channels pass cesium ions with a single-channel conductance of ∼21pS. Although Cav1.2 opening events were best described kinetically with two open time constants, Cav1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Cav1.4 channels by ∼45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Cav1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.

Activation of transient receptor potential vanilloid 1 (TRPV1) by resiniferatoxin

Transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+) permeable non-selective cation channel activated by physical and chemical stimuli. Resiniferatoxin (RTX), an ultrapotent agonist of TRPV1, is under investigation for treatment of urinary bladder hyper-reflexia and chronic pain conditions. Here, we have determined the characteristics of RTX-induced responses in cells expressing native and cloned rat TRPV1. Whole-cell currents increase with repeated application of submaximal concentrations of RTX until a maximal response is attained and do not deactivate even after prolonged washout. Interestingly, the rate of activation and block by capsazepine of RTX-induced currents are significantly slower than for capsaicin-induced currents. RTX-induced whole-cell currents are outwardly rectifying, but to a lesser extent than capsaicin-induced currents. RTX-induced single channel currents exhibit multiple conductance states and outward rectification. The open probability (P(o)) of RTX-induced currents is higher at all potentials as compared to capsaicin-induced currents, which showed a strong voltage-dependent decrease at negative potentials. Single-channel kinetic analyses reveal that open-time distribution of RTX-induced currents can be fitted with three exponential components at negative and positive potentials. The areas of distribution of the longer open time constants are significantly larger than capsaicin-induced currents. The closed-time distribution of RTX-induced currents can be fitted with three exponential components as compared to capsaicin-induced currents, which require four exponential components. Current-clamp experiments reveal that low concentrations of RTX caused a slow and sustained depolarization beyond threshold while generating few action potentials. Concentrations of capsaicin required for the same extent of depolarization generated a significantly greater number of action potentials. These properties of RTX may play a role in its clinical usefulness.

Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac K(ATP) channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the K(ATP) channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2delta C26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac K(ATP) channel, 5 microM 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2delta C26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.