Biexponential Time Course Research Articles

Zinc pyrithione activates K+ channels and hyperpolarizes the membrane of rat pulmonary artery smooth muscle cells.

The membrane potential helps determine pulmonary artery smooth muscle cell (PASMC) contraction. The Kv7 channel activators, retigabine and flupirtine, are thought to dilate pulmonary arteries by hyperpolarising PASMC. Zinc pyrithione activates Kv7 channels by a mechanism distinct from retigabine and with different Kv7 subunit selectivity. This study aimed to determine if zinc pyrithione selectively activates Kv7 channels in rat PASMC to evoke pulmonary artery dilation. Zinc pyrithione relaxed pulmonary arteries with half-maximal effect at 4.3μM. At 10μM it activated pronounced voltage-dependent K+ current and hyperpolarized PASMCs by around 10mV. Tetraethylammonium ions (TEA, 10mM) and paxilline (1μM) abolished both the current and hyperpolarisation. XE991 (10μM) blocked the hyperpolarization and reduced the current by 30%. Iberiotoxin (50nM) had no effect on the hyperpolarisation, but reduced the current by 40%. The XE991-sensitive current activated with an exponential time course (time constant 17ms), whereas the iberiotoxin-sensitive current followed a bi-exponential time course (time constants 6 and 57ms), suggesting that the drugs blocked different components of the zinc pyrithione-induced current. Zinc pyrithione therefore appears to activate at least two types of K+ channel in PASMC; an XE991, TEA and paxilline-sensitive Kv7 channel and a TEA, paxilline and iberiotoxin-sensitive BKCa channel. Both could contribute to the relaxing effect of zinc pyrithione on pulmonary artery.

Kinetics of Inhibitory Feedback from Horizontal Cells to Photoreceptors: Implications for an Ephaptic Mechanism.

Inhibitory feedback from horizontal cells (HCs) to cones generates center-surround receptive fields and color opponency in the retina. Mechanisms of HC feedback remain unsettled, but one hypothesis proposes that an ephaptic mechanism may alter the extracellular electrical field surrounding photoreceptor synaptic terminals, thereby altering Ca(2+) channel activity and photoreceptor output. An ephaptic voltage change produced by current flowing through open channels in the HC membrane should occur with no delay. To test for this mechanism, we measured kinetics of inhibitory feedback currents in Ambystoma tigrinum cones and rods evoked by hyperpolarizing steps applied to synaptically coupled HCs. Hyperpolarizing HCs stimulated inward feedback currents in cones that averaged 8-9 pA and exhibited a biexponential time course with time constants averaging 14-17 ms and 120-220 ms. Measurement of feedback-current kinetics was limited by three factors: (1) HC voltage-clamp speed, (2) cone voltage-clamp speed, and (3) kinetics of Ca(2+) channel activation or deactivation in the photoreceptor terminal. These factors totaled ∼4-5 ms in cones meaning that the true fast time constants for HC-to-cone feedback currents were 9-13 ms, slower than expected for ephaptic voltage changes. We also compared speed of feedback to feedforward glutamate release measured at the same cone/HC synapses and found a latency for feedback of 11-14 ms. Inhibitory feedback from HCs to rods was also significantly slower than either measurement kinetics or feedforward release. The finding that inhibitory feedback from HCs to photoreceptors involves a significant delay indicates that it is not due to previously proposed ephaptic mechanisms. Lateral inhibitory feedback from horizontal cells (HCs) to photoreceptors creates center-surround receptive fields and color-opponent interactions. Although underlying mechanisms remain unsettled, a longstanding hypothesis proposes that feedback is due to ephaptic voltage changes that regulate photoreceptor synaptic output by altering Ca(2+) channel activity. Ephaptic processes should occur with no delay. We measured kinetics of inhibitory feedback currents evoked in photoreceptors with voltage steps applied to synaptically coupled HCs and found that feedback is too slow to be explained by ephaptic voltage changes generated by current flowing through continuously open channels in HC membranes. By eliminating the proposed ephaptic mechanism for HC feedback regulation of photoreceptor Ca(2+) channels, our data support earlier proposals that synaptic cleft pH changes are more likely responsible.

A rapid form of activity-dependent recovery from short-term synaptic depression in the intensity pathway of the auditory brainstem

Short-term synaptic plasticity acts as a time- and firing rate-dependent filter that mediates the transmission of information across synapses. In the avian auditory brainstem, specific forms of plasticity are expressed at different terminals of the same auditory nerve fibers and contribute to the divergence of acoustic timing and intensity information. To identify key differences in the plasticity properties, we made patch-clamp recordings from neurons in the cochlear nucleus responsible for intensity coding, nucleus angularis, and measured the time course of the recovery of excitatory postsynaptic currents following short-term synaptic depression. These synaptic responses showed a very rapid recovery, following a bi-exponential time course with a fast time constant of approximately 40 ms and a dependence on the presynaptic activity levels, resulting in a crossing over of the recovery trajectories following high-rate versus low-rate stimulation trains. We also show that the recorded recovery in the intensity pathway differs from similar recordings in the timing pathway, specifically the cochlear nucleus magnocellularis, in two ways: (1) a fast recovery that was not due to recovery from postsynaptic receptor desensitization and (2) a recovery trajectory that was characterized by a non-monotonic bump that may be due in part to facilitation mechanisms more prevalent in the intensity pathway. We tested whether a previously proposed model of synaptic transmission based on vesicle depletion and sequential steps of vesicle replenishment could account for the recovery responses, and found it was insufficient, suggesting an activity-dependent feedback mechanism is present. We propose that the rapid recovery following depression allows improved coding of natural auditory signals that often consist of sound bursts separated by short gaps.

A model of endocannabinoid 2-AG-mediated depolarization-induced suppression of inhibition

Depolarization-induced suppression of inhibition (DSI) is known to be mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG). It's calcium-dependent production and subsequent retrograde diffusion from postsynaptic pyramidal cells to presynaptic cannabinoid receptors (CB1) located on the preterminal axon and the perisynapse of CCK-containing basket cells in the CA1 region of the hippocampus transiently suppresses GABA transmitter release [1,3]. This endocannabinoid signaling system has many physiological implications for memory-related synaptic plasticity and gamma oscillations; exogenous application of synthetic cannabinoids has a dramatic impact on the functioning of this system, more precisely the loss of synchronized cell assemblies by modification of inhibitory feedback loops in the CA1 region of the hippocampus [4,5], thus altering outcome of spatial memory-related tasks in animals and learning in humans. This study uses computational modeling methods to understand the modes of production and action of the endocannabinoid 2-AG in monosynaptically suppressing transmitter release. Using the NEURON simulation environment [2], we developed an isopotential model cell with an L-type Ca2+ channel being the sole pathway for Ca2+ entry required for synthesis of 2-AG. The observed time courses for 2-AG passive diffusion (between adjacent shell compartments), and uptake and intracellular hydrolysis can be observed with successive depolarizing current pulses delivered by a single-electrode voltage clamp. These changes in concentration levels in the compartment representing the perisynapse and preterminal axon (the CCK-positive basket area covering the pyramidal cell expressing CB1) are directly proportional to the GABA synaptic depression due to inhibition of presynaptic calcium channels. The amount of CB1 activation is described by the Langmuir equation and, together with the Ca2+ ion cooperativity for transmitter release, affects the synaptic conductance profile described by the solution of two coupled linear ODEs. It is shown that in the model, [Ca2+]i had to rise to 0.1μM in order to sufficiently activate the kinetic model describing the Phospholipase C-Diacylglycerol pathway (PLC-DAG) for 2-AG production [1,3]. We also show highly simplified dynamics of PLC after stimulation by [Ca2+]i , DAG production, and the spatial gradient in 2-AG across compartments (discretization of Fick's second law for diffusion). Uptake and intracellular hydrolysis is driven by the concentration gradient and the association rate constant for the irreversible reaction hydrolysing 2-AG. The end result observed is the recovery of biexponential postsynaptic conductance time course, which can vary depending on as yet unknown parameter values. The suppression can be effective over a much smaller timescale due to summation of high-frequency inputs by the synapse.

A Modeling Study of T-Type Ca 2+ Channel Gating and Modulation by L-Cysteine in Rat Nociceptors

L-cysteine (L-cys) increases the amplitude of T-type Ca 2+ currents in rat T-rich nociceptor-like dorsal root ganglia neurons. The modulation of T-type Ca 2+ channel gating by L-cys was studied by fitting Markov state models to whole-cell currents recorded from T-rich neurons. The best fitting model tested included three resting states and inactivation from the second resting state and the open state. Inactivation and the final opening step were voltage-independent, whereas transitions between the resting states and deactivation were voltage-dependent. The transition rates between the first two resting states were an order of magnitude faster than those between the second and third resting states, and the voltage-dependency of forward transitions through resting states was two to three times greater than for analogous backward transitions. Analysis with the best fitting model suggested that L-cys increases current amplitude mainly by increasing the transition rate from resting to open and decreasing the transition rate from open to inactivated. An additional model was developed that could account for the bi-exponential time course of recovery from inactivation of the currents and the high frequency of blank sweeps in single channel recordings. This model detected basically the same effects of L-cys on channel gating as the best fitting model.

Fast changes in the functional status of release sites during short‐term plasticity: involvement of a frequency‐dependent bypass of Rac at Aplysia synapses

Synaptic transmission can be described as a stochastic quantal process defined by three main parameters: N, the number of functional release sites; P, the release probability; and Q, the quantum of response. Many changes in synaptic strength that are observed during expression of short term plasticity rely on modifications in P. Regulation of N has been also suggested. We have investigated at identified cholinergic inhibitory Aplysia synapses the cellular mechanism of post-tetanic potentiation (PTP) expressed under control conditions or after N has been depressed by applying lethal toxin (LT) from Clostridium sordellii or tetanus toxin (TeNT). The analysis of the Ca(2+) dependency, paired-pulse ratio and variance to mean amplitude relationship of the postsynaptic responses elicited at distinct extracellular [Ca(2+)]/[Mg(2+)] elicited during control post-tetanic potentiation (PTP(cont)) indicated that PTP(cont) is mainly driven by an increase in release probability, P. The PTP expressed at TeNT-treated synapses (PTP(TeNT)) was found to be similar to PTP(cont), but scaled to the extent of reduction in N produced by TeNT. Despite LT inducing a decrease in N as TeNT does, the PTP expressed at LT-treated synapses (PTP(LT)) was characterized by exceptionally large amplitude and bi-exponential time course, as compared to PTP(cont) or the PTP(TeNT). Analysis of the Ca(2+) dependency of PTP(LT), paired-pulse ratio and fluctuations in amplitude of the postsynaptic responses elicited during PTP(LT) or the variance to mean amplitude relationship of time-locked postsynaptic responses in a series of subsequent PTP(LT) indicated that an N-driven change is involved in the early phase (1 s time scale) of PTP(LT), while at a later stage PTP(LT) is composed of both N and P increases. Our results suggest that fast switching on of the functional status of the release sites occurs also during the early events of PTP(cont). The early N-driven phase of PTP(LT) is likely to be a functional recovery of the release sites silenced by Rac inactivation. This effect did not appear to result from reversion of LT inhibitory action but from bypassing the step regulated by Rac. Altogether the data suggest that Rac and the intracellular pathway which allows the bypassing of Rac are key players in new forms of short-term plasticity that rely on fast, activity-dependent changes in the functional status of the release sites.

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.

Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel.

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (Vm) and time dependence of the protopore (Pf) and common (Ps) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (ICl) were measured using whole cell patch clamp. WT channels had ICl that showed inward rectification and biexponential time course. Time constants of fast and slow components were ∼10-fold different at negative Vm and corresponded to Pf and Ps, respectively. Pf and Ps were ∼1 at −200 mV, while at Vm ≥ 0 mV, Pf ∼ 0 and Ps ∼ 0.6. Hence, Pf dominated open kinetics at moderately negative Vm, while at very negative Vm both gates contributed to gating. At Vm ≥ 0 mV, mClC-2 closes by shutting off Pf. Three- and two-state models described the open-to-closed transitions of Pf and Ps, respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter Pf or Ps in other ClC channels. Based on the time and Vm dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to Pf, the dominant regulator of gating, while the C258 residue alters the Vm dependence of Pf, probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

Membrane transport of anandamide through resealed human red blood cell membranes

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments at 0 degrees C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 +/- 0.023, and the rate constant of unidirectional flux from inside to outside is 0.361 +/- 0.023 s(-1). The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]o) increases with the square root of [BSA]o in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane very rapidly, within seconds. At a molar ratio of anandamide to BSA of <1, membrane binding of anandamide increases with increasing temperatures between 0 degrees C and 37 degrees C, and the equilibrium dissociation constants are in the nanomolar range. The nature of membrane binding and the mechanism of membrane translocation are discussed.

Single-channel recordings of a rapid delayed rectifier current in adult mouse ventricular myocytes: basic properties and effects of divalent cations.

The rapidly delayed rectifier current (I(Kr)) has been described in ventricular myocytes isolated from many species, as well as from neonatal mice. However, whether I(Kr) is present in the adult mouse heart remains controversial. We used cell-attached patch-clamp recording in symmetrical K(+) solutions to assess the presence and behaviour of single I(Kr) channels in adult mouse cardiomyocytes (mI(Kr)). Of 314 patches, 158 (50.1%) demonstrated mI(Kr) currents as compared with 131 (42.3%) for the I(K1) channel. Single mI(Kr) channel activity was rarely observed at potentials positive to -10 mV. The slope conductance at negative potentials was 12 pS. Upon repolarization, ensemble-averaged mI(Kr) showed slow deactivation with a biexponential time course. A selective I(Kr) blocker, E-4031 (1 microm), completely blocked mI(Kr) channel activity. Extracellular Ca(2+) and Mg(2+) at physiological concentrations shifted the activation by approximately 30 mV, accelerated deactivation kinetics, prolonged long-closed time, and reduced open probability without affecting single-channel conductance, suggesting a direct channel-blocking effect in addition to well-recognized voltage shifts. HERG subunits expressed in Chinese hamster ovary cells produced channels with properties similar to those of mI(Kr), except for the more-negative activation of the HERG channels. Despite the abundant expression of mI(Kr), single-channel events were rarely observed during action-potential clamp and 5 microm E-4031 had no detectable effect on the action potential parameters, confirming that mI(Kr) plays at best a minor role in repolarization of adult mouse cardiomyocytes, probably because the modulatory effects of divalent cations prevent significant mI(Kr) opening under physiological conditions.

Kinetic properties of Kv4.3 and their modulation by KChIP2b

KChIPs are a family of Kv4 K + channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20 s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K + channels.

The speeding of EPSC kinetics during maturation of a central synapse.

Several factors contribute to the shape of excitatory postsynaptic currents (EPSCs) in CNS neurons, among them the kinetics of presynaptic release, transmitter clearance, and the properties and distribution of postsynaptic receptors. The decays of AMPA receptor-mediated EPSCs at rat cerebellar mossy fibre-granule cell (MF-gc) synapses follow a bi-exponential time-course. The fast component dominates the decay, accounting for 84-94% of the peak amplitude. Here we show that both components of decay, and also the risetimes, became faster during postnatal maturation. At adult, but not immature, synapses, the risetimes and decays of evoked multiquantal EPSCs were similar to those of monoquantal miniature (m)EPSCs. The faster risetimes at mature synapses reflected increased synchrony of multivesicular release, whereas the faster decays appeared to reflect changes in the properties of postsynaptic receptors. Inhibition of glutamate uptake was without effect on evoked EPSCs at both ages. Furthermore, after slowing receptor desensitization with cyclothiazide, the EPSCs at mature synapses decayed as slowly as EPSCs at immature synapses, suggesting that faster glutamate clearance does not account for the developmental speeding of EPSC decay. Our results support previous conclusions that glutamate clearance and receptor deactivation are important determinants of the fast decay component at immature synapses. Desensitization becomes increasingly important during development and plays a major role in shaping EPSC decay at mature synapses.

Synaptojanin 1 Contributes to Maintaining the Stability of GABAergic Transmission in Primary Cultures of Cortical Neurons

Inhibitory synapses in the CNS can exhibit a considerable stability of neurotransmission over prolonged periods of high-frequency stimulation. Previously, we showed that synaptojanin 1 (SJ1), a presynaptic polyphosphoinositide phosphatase, is required for normal synaptic vesicle recycling (Cremona et al., 1999). We asked whether the stability of inhibitory synaptic responses was dependent on SJ1. Whole-cell patch-clamp recordings of unitary IPSCs were obtained in primary cortical cultures between cell pairs containing a presynaptic, fast-spiking inhibitory neuron (33.5-35 degrees C). Prolonged presynaptic stimulation (1000 stimuli, 2-20 Hz) evoked postsynaptic responses that decreased in size with a bi-exponential time course. A fast component developed within a few stimuli and was quantified with paired-pulse protocols. Paired-pulse depression (PPD) appeared to be independent of previous GABA release at intervals of >/=100 msec. The characteristics of PPD, and synaptic depression induced within the first approximately 80 stimuli in the trains, were unaltered in SJ1-deficient inhibitory synapses. A slow component of depression developed within hundreds of stimuli, and steady-state depression showed a sigmoidal dependence on stimulation frequency, with half-maximal depression at 6.0 +/- 0.5 Hz. Slow depression was increased when release probability was augmented, and there was a small negative correlation between consecutive synaptic amplitudes during steady-state depression, consistent with a presynaptic depletion process. Slow depression was increased in SJ1-deficient synapses, with half-maximal depression at 3.3 +/- 0.9 Hz, and the recovery was retarded approximately 3.6-fold. Our studies establish a link between a distinct kinetic component of physiologically monitored synaptic depression and a molecular modification known to affect synaptic vesicle reformation.

Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog

Changes in cytosolic free Ca2+concentration ([Ca2+]i) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca2+-selective dye Oregon Green 488 BAPTA-1 (OG, 100μM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca2+entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca2+]iat individual hotspots rose with a time constant τ1∼70ms and decayed with a bi-exponential time-course (τ2∼160, τ3∼2500ms) following a 160ms depolarization to −20mV. With repeated stimulation [Ca2+]iunderwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca2+channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca2+channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1s), Ca2+was able to reach the cell apical ciliated pole. The effective Ca2+diffusion constant, measured from the progression of Ca2+wavefronts in the cytosol, was ∼57mmμm2/s. Our results indicate that in these hair cells, buffered diffusion of Ca2+proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers.

Effects of BDF 9198 on action potentials and ionic currents from guinea-pig isolated ventricular myocytes.

BDF 9198 (a congener of DPI 201 - 106 and BDF 9148) was found to be a positive inotrope on guinea-pig isolated ventricular muscle strips. The effects of BDF 9198 on action potentials and ionic currents from guinea-pig isolated ventricular myocytes were studied using the whole cell patch clamp method. In normal external solution, at 37 degrees C, action potential duration at 50% repolarization (APD(50)) was 167.4+/-8.36 ms (n=37). BDF 9198 produced a concentration-dependent increase in APD(50) (no significant increase at 1x10(-10) M; and APD(50) values of 273.03+/-35.8 ms at 1x10(-9) M; n=6, P<0.01 and 694.7+/-86.3 ms at 1x10(-7) M; P<0.001, n=7). At higher concentrations in the range tested, BDF 9198 also induced early and delayed and after-depolarizations. Qualitative measurements of I(Na) with physiological [Na](o) showed prolongation of the current by BDF 9198, and the appearance of transient oscillatory inward currents at high concentrations. Quantitative recording conditions for I(Na) were established using low external [Na] and by making measurements at room temperature. The current - voltage relation, activation parameters and time-course of I(Na) were similar before and after a partial blocking dose of Tetrodotoxin (TTX, 1 microM), despite a 2 fold difference in current amplitude. This suggests that voltage-clamp during flow of I(Na) was adequately maintained under our conditions. Selective measurements of I(Na) at room temperature showed that BDF 9198 induced a concentration-dependent, sustained component of I(Na) (I(Late)) and caused a slight left-ward shift in the current - voltage relation for peak current. The drug-induced I(Late) showed a similar voltage dependence to peak current in the presence of BDF 9198. Both peak current and I(Late) were abolished by 30 microM TTX and were sensitive to external [Na]. Inactivation of control I(Na) during a 200 ms test pulse to -30 mV followed a bi-exponential time-course. In addition to inducing a sustained current component, BDF 9198 left the magnitude of the fast inactivation time-constant unchanged, but increased the magnitude of the slow inactivation time-constant. Additional experiments with a longer pulse (1 s) raised the possibility that in the presence of BDF 9198, I(Na) inactivation may be comprised of more than two phases. No significant effects of 1x10(-6) M BDF 9198 were observed on the L-type calcium current, or delayed and inward rectifying potassium currents measured at 37 degrees C. It is concluded that the prolongation of APD(50) by BDF 9198 resulted from selective modulation of I(Na). Reduced current inactivation induced a persistent I(Na), increasing the net depolarizing current during the action potential. This action of the drug indicates a potential for 'QT prolongation' of the ECG. The observation of after-depolarizations suggests a potential for proarrhythmia at some drug concentrations.

A blocker-resistant, fast-decaying, intermediate-threshold calcium current in palaeocortical pyramidal neurons.

The whole-cell patch-clamp technique was used to record Ca2+ currents in acutely dissociated neurons from layer II of guinea-pig piriform cortex (PC). Ba2+ (5 mM) was used as charge carrier. In a subpopulation of layer II cells ( approximately 22%) total Ba2+ currents (IBas) displayed a high degree (> 70%) of inactivation after 300 ms of steady depolarization. The application of L-, N- and P/Q-type Ca2+-channel blockers to these high-decay IBas left their fast inactivating component largely unaffected. The inactivation phase of the blocker-resistant, fast-decaying IBa thus isolated had a bi-exponential time course, with a fast time constant of approximately 20 ms and a slower time constant of approximately 100 ms at voltage levels positive to -10 mV. The voltage dependence of activation of the blocker-resistant, fast-decaying IBa was shifted by approximately 7-9 mV in the negative direction in comparison with those of other pharmacologically and/or kinetically different high-voltage-activated Ca2+ currents. We named this blocker-resistant, fast-decaying, intermediate-threshold current IRfi. The amplitude of IRfi decreased only slightly (by approximately 9%) when extracellular Ca2+ was substituted for Ba2+, in contrast with that of slowly decaying, high-voltage-activated currents, which was reduced by approximately 41% on average. Moreover, IRfi was substantially inhibited by low concentrations of Ni2+ (50 microM). We conclude that IRfi, because of its fast inactivation kinetics, intermediate threshold of activation and resistance to organic blockers, represents a definite, identifiable Ca2+ current different from classical high-voltage-activated currents and clearly distinguishable from classical IT. The striking similarity found between IRfi and Ca2+ currents resulting from heterologous expression of alpha1E-type channel subunits is discussed.

Identification of a T-Type Ca 2+ Channel Isoform in Murine Atrial Myocytes (AT-1 Cells)

Calcium channels are important targets for therapeutics, but their molecular diversity complicates characterization of these channels in native heart cells. In this study, we identify a new splice variant of a low-voltage activated, or T-type Ca(2+), channel in murine atrial myocytes. To date, alpha1G and alpha1H are the only 2 T-type Ca(2+) channel isoforms found in cardiovascular tissue. We compared alpha1G and alpha1H channel current heterologously expressed in HEK 293 cells with T-type current from the murine atrial tumor cell, AT-1. AT-1 cell T-type current (I(T)) has the same voltage dependence of activation and inactivation as alpha1G and alpha1H. The cloned T-type channels and AT-1 T-type current share similar kinetics of macroscopic inactivation and deactivation. The kinetics of recovery from inactivation of T-type currents serves as an electrophysiological signature for T-channel isoform. alpha1G and AT-1 I(T) have a similar recovery from inactivation time course that is faster than that for alpha1H. In all cases, T-type current recovers with a biexponential time course, and the relative amplitude of fast and slow time courses explains the slower alpha1H recovery kinetics, rather than differences in the time constants of the individual transitions. Thus, the T-type channels may be an important contributor to automaticity in heart cells, and molecular diversity is reflected in the pathway of recovery from inactivation.

A Novel Anionic Inward Rectifier in Native Cardiac Myocytes

Although the cationic inward rectifiers (Kir and hyperpolarization-activated I(f) channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl(-)) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (-40 to -140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7+/-23.4 [mean+/-SEM] and 2073.6+/-287.6 ms at -120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl(-) (I(Cl.ir)) and had an anion permeability sequence of Cl(-)>I(-)>>aspartate. I(Cl.ir) was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar I(Cl.ir) was also observed in guinea pig cardiac myocytes. The properties of I(Cl.ir) are consistent with currents generated by expression of ClC-2 Cl(-) channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel I(Cl.ir) is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions.

Mechanism and Cellular Applications of a Green Fluorescent Protein-based Halide Sensor

We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the absence of Cl(-), which increased to 7.86 at 150 mM Cl(-). At pH 7.5, YFP-H148Q fluorescence decreased maximally by approximately 2-fold with a K(D) of 100 mM Cl(-). YFP-H148Q had a fluorescence lifetime of 3.1 ns that was independent of pH and [Cl(-)]. Circular dichroism and absorption spectroscopy revealed distinct Cl(-)-dependent spectral changes indicating Cl(-)/YFP binding. Stopped-flow kinetic analysis showed a biexponential time course of YFP-H148Q fluorescence (time constants <100 ms) in response to changes in pH or [Cl(-)], establishing a 1:1 YFP-H148Q/Cl(-) binding mechanism. Photobleaching analysis revealed a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers. The anion selectivity sequence for YFP-H148Q quenching (ClO(4)(-) approximately I(-) > SCN(-) > NO(3)(-) > Cl(-) > Br(-) > formate > acetate) indicated strong binding of weakly hydrated chaotropic ions. The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the tri-amino acid chromophore. YFP-H148Q transfected mammalian cells were brightly fluorescent with cytoplasmic/nuclear staining. Ionophore calibrations indicated similar YFP-H148Q pH and anion sensitivities in cells and aqueous solutions. Cyclic AMP-regulated Cl(-) transport through plasma membrane cystic fibrosis transmembrane conductance regulator Cl(-) channels was assayed with excellent sensitivity from the time course of YFP-H148Q fluorescence in response to extracellular Cl(-)/I(-) exchange. The green fluorescent protein-based halide sensor described here should have numerous applications, such as anion channel cloning by screening of mammalian expression libraries and discovery of compounds that correct the cystic fibrosis phenotype by screening of combinatorial libraries.

Permeation and Gating Properties of the Novel Epithelial Ca2+ Channel

The recently cloned epithelial Ca(2+) channel (ECaC) constitutes the Ca(2+) influx pathway in 1,25-dihydroxyvitamin D(3)-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca(2+) concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca(2+) gradient, demonstrating the distinctive Ca(2+) permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below -40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba(2+) or Sr(2+) replaced Ca(2+) and was absent in Ca(2+)-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P(Ca):P(Na) calculated from the reversal potential at 30 mM [Ca(2+)](o) was larger than 100. The divalent cation selectivity profile is Ca(2+) > Mn(2+) > Ba(2+) approximately Sr(2+). Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca(2+) was replaced by Ba(2+) and was virtually abolished if [Ca(2+)](o) was lowered to 1 nM. In conclusion, ECaC is a Ca(2+) selective channel, exhibiting Ca(2+)-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.