Instantaneous Current-voltage Relationship Research Articles

Instantaneous current-voltage relationships during the course of the human cardiac ventricular action potential: new computational insights into repolarization dynamics.

To adopt a novel three-dimensional (3D) representation of cardiac action potential (AP) to compactly visualize dynamical properties of human cellular ventricular repolarization. We have recently established a novel 3D representation of cardiac AP, which is based on the iterative measurement of instantaneous ion current-voltage profiles during the course of an AP. Such an approach has been originally developed on real patch-clamped ventricular cells, and subsequently improved in silico on several cardiac ventricular AP models of different mammals, and on models of different AP types of the human heart. We apply it here on two different models of human ventricular AP, and show that it compactly provides further insights into repolarization dynamics. The 3D representation of the AP includes equilibrium points during repolarization, and can be screened in terms of what we have shown to be a region, during late repolarization, when membrane conductance becomes negative and repolarization therefore auto-regenerative. We have called this time window auto-regenerative-repolarization-phase (ARRP). In addition to previous findings obtained through the same procedure, we show here that 3D current-voltage-time representations of human ventricular AP allow compact visualization of dynamical properties, which are relevant for the physiology and pathology of ventricular repolarization. In particular, we suggest that the volume under the current surface corresponding to the ARRP might be used as a predictor of safety of repolarization, in single cells and during AP conduction in cell pairs.

A protocol combining current- and voltage-clamp provides a novel and useful three-dimensional representation of cardiac action potential

A compact three-dimensional representation of cardiac action potential (AP) properties in terms of current source is presented here. The experimental protocol used to obtain such representation is based on the measure of instantaneous current-voltage relationships during the course of the AP. The procedure, which combines current- and voltage-clamps on patch clamped cardiac myocytes, has been previously applied to real cells, and then extended to computer simulations with cellular ventricular AP models. The three-dimensional AP representation allows to easily estimate membrane resistance during repolarization, a key factor for the modulation of ventricular repolarization. It also shows that, during late ventricular repolarization, membrane conductance becomes negative, <em>i.e.</em> repolarization is auto-regenerative. The novel AP representation is therefore a useful tool for both in vivo and in silico cardiac cellular electrophysiological investigations.

Динамічні електричні стани неоднорідних популяцій іонних каналів у мембранах збудливих клітин

In computer models, we studied instantaneous (time-varying) current-voltage relationships (iIVs) of populations of ion channels characteristic of the membrane of different type excitable cells, of which the responses to electrical stimuli essentially differ: giant squid axon (Hodgkin-Huxley model), cardiomyocyte, dendrites of CA3 hippocampal pyramidal neurons and Purkinje neurons of the cerebellum. The membrane potential was stepped from the rest level to a certain depolarization test level that was clamped for a certain time, and the total current was measured at different moments after the step onset. For each iIV zero-current points (potentials) were determined. A set of such points, which were situated on the limb of iIV positive slop and corresponded to the state of high membrane depolarization (excitation state, upstate) at different time moments, were used to characterize the dynamics of the excitation state in time. With these indicators the axon membrane was characterized by a single excitation state that rapidly occurred (0.25 ms) and was short-lasting (decayed from -45 to 40 mV during life-time of 5.5 ms). There were two such states of the membrane of cardiomyocyte. The first one was early, rapidly occurring and short-living (rapidly relaxing). It occurred shortly after the depolarization start and lasted for 14.5 ms. The second one was late, slowly rising and long-lasting (occurred with a 7.5-ms delay, increased from 11 to 46 mV in 39 ms and then relaxed lasting for 623 ms in total). The dendritic membrane ofCA3 neurons had one long-lasting excitation state that occurred shortly after the depolarization shift, first rapidly relaxed during 3 ms from initial 30 mV level to -10 mV and then slowly, in 80 ms, stabilized at the level of -20 mV. In the Purkinje neuron membrane two short-lasting and one very long-lasting excitation states were revealed. The first state of very high (>100 mV) depolarization relaxed to 4 mV in 0.8 ms. Shortly before its vanishing, at 0.7 ms, the second short-lasting state emerged, which relaxed in 1 ms from -22 mV to -48 mV. At 1.8 ms a new excitation state emerged, which after a transient relaxation stabilized at -29.65 mV starting from 88 ms. Thus, iIVs allowed disclosing a fine organization of the states of electrical excitation of the membrane and revealing, in populations of ion channels of different content, existence of different number of the mentioned states, which differ from each other in occurrence time and life-time.

Heterogeneity of Intrinsic Repolarization Properties Within the Human Heart: New Insights From Simulated Three-Dimensional Current Surfaces

Heterogeneity of repolarization properties is pivotal for both physiology and pathology of the heart and mathematical models of different cardiac cell types that are tuned to experimental data in order to reproduce it in silico. Repolarization heterogeneity is described most of the times with reference to one or the other of the many repolarization parameters, like action potential (AP) form and duration, or the maximum conductance of a given ion current, which are nonlinearly connected and frequently overdetermined. A compact representation of models dynamics would help their standardization, their use, and the understanding of the underlying physiology. A 3-D representation of cardiac AP derived from the measure of instantaneous current-voltage relationships during repolarization has been previously described. Here, it is shown that such a representation compactly summarizes important features of repolarization which are relevant particularly for what concerns its electrotonic modulation within the human heart. It is found that, according to the tested models, late phase of AP repolarization displays autoregenerativity only within the ventricle, and that this property is heterogeneously distributed across the wall. Three-dimensional current representations of the AP also provide precise estimation of the time course of membrane resistance, which changes throughout the heart, and can be used to predict entrainment of repolarization during AP propagation.

Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood-brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87-MG cells migration. Using the perforated patch-clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM-34 sensitive, intermediate-conductance calcium-activated K (IK(Ca) ) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS- and NPPB-sensitive Cl current displaying an outward rectifying instantaneous current-voltage relationship and a slow, voltage-dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IK(Ca) current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS-induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM-34 and DIDS on the FCS-induced U87-MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87-MG cell migration in the presence of FCS, and that their co-application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS-induced glioblastoma cell migration.

Evaluation of a Two-Site, Three-Barrier Model for Permeation in CaV3.1 (α1G) T-Type Calcium Channels: Ca2+, Ba2+, Mg2+, and Na+

We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide range (100 nM: -110 mM: ) while recording whole-cell currents over a wide voltage range (-150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current-voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca(2+) and Ba(2+), and shifts in reversal potential between Ca(2+) and Ba(2+). The fit to block by 1 mM Mg(2+)(i) was reasonable, but block by Mg(2+)(0) was described less well. Surprisingly, fits were comparable with strong ion-ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba(2+) preferred the inner site, while Ca(2+) preferred the outer site, potentially explaining faster entry of Ni(2+) and other pore blockers when Ba(2+) is the charge carrier.

Permeation and Gating in CaV3.1 (α1G) T-type Calcium Channels Effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through CaV3.1 T-type calcium channels, varying Ca2+ and Ba2+ over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current–voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by PCa/PNa = 87 and PCa/PBa = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent Kd values were similar for Ca2+ and Ba2+, both for block of currents carried by Na+ (3 μM for Ca2+ vs. 4 μM for Ba2+, at −30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca2+ vs. 2.5 mM for Ba2+; nearly voltage independent). Block by 3–10 μM Ca2+ was time dependent, described by bimolecular kinetics with binding at ∼3 × 108 M−1s−1 and voltage-dependent exit. Ca2+o, Ba2+o, and Mg2+o also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e− per 98 Å2 from Gouy-Chapman theory. Additionally, inward currents inactivated ∼35% faster in Ba2+o (vs. Ca2+o or Na+o). The accelerated inactivation in Ba2+o correlated with the transition from Na+ to Ba2+ permeation, suggesting that Ba2+o speeds inactivation by occupying the pore. We conclude that the selectivity of the “surface charge” among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca2+ or Ba2+.

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg 2+ concentration on L-type Ca 2+ ( I Ca) and Ba 2+ currents ( I Ba) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8 mM [Mg 2+] ([Mg 2+] p) buffered with 30 mM citrate and 10 mM ATP. Increasing [Mg 2+] p from 0.2 to 1.8 mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current–voltage relationships were studied with two-pulse and tail current ( I T) protocols, respectively. Increasing [Mg 2+] p shifted the V M for half inactivation by −20 mV but dramatically decreased I Ca amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I T amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg 2+] p might also inhibit permeation through open Ca 2+ channels at positive V M.

Voltage Dependence of the Ca2+-activated Cation Channel TRPM4

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.

Sodium blocking induced by a point mutation at the C-terminal end of the pore helix of the KAT1 channel.

A plant hyperpolarization-activating K+ channel, KAT1, is highly selective for K+ over Na+ and is little affected by external Na+, which is crucial to take up K+ effectively in a Na+-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na+. We report here electrophysiological experiments for the mechanism of action of external Na+ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na+. However, the activity of both mutant channels was significantly depressed by Na+ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na+ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na+ to penetrate into the pore.

GABA-evoked chloride currents do not differ between dendrites and somata of rat neocortical neurons.

1. We performed patch-clamp recordings on acutely isolated somata and dendritic segments of rat neocortical neurons, in order to compare the reversal potential (E(GABA)) and relative density of GABA(A) receptor-mediated Cl(-) currents in these two cellular compartments. 2. Currents were recorded with the Cl(-)-impermeable pore former gramicidin (25--75 microg ml(-1)) in HCO(3)(-)-free bath solution. Voltage ramps (-110 to -30 mV) from a holding potential (V(h)) of -60 mV in the absence and presence of 2 microM GABA were used to construct instantaneous current-voltage relationships. Currents were abolished by co-application of GABA with the GABA(A) receptor antagonist bicuculline (40 microM). 3. GABA conductance, normalized to membrane surface area, was not different in somata and dendrites. In addition, E(GABA) was not different in the two compartments. 4. Replacement of intracellular K(+) with Cs(+) resulted in a significantly more depolarized E(GABA) in both somata and dendrites. These results suggest that the resting intracellular Cl(-) concentration ([Cl(-)](i)) is similar in somata and dendrites and that an outward Cl(-) transporter system maintains low [Cl(-)](i).

KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology.

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than -60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of -32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC(50) values of 5.5 and 14 microM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC(50) value of 9.4 microM, whereas clofilium was without significant effect at 100 microM. The KCNQ4-expressing cells exhibited average resting membrane potentials of -56 mV in contrast to -12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.

Participation of a chloride conductance in the subthreshold behavior of the rat sympathetic neuron.

The presence of a novel voltage-dependent chloride current, active in the subthreshold range of membrane potential, was detected in the mature and intact rat sympathetic neuron in vitro by using the two-microelectrode voltage-clamp technique. Hyperpolarizing voltage steps applied to a neuron held at -40/-50 mV elicited inward currents, whose initial magnitude displayed a linear instantaneous current-voltage (I-V) relationship; afterward, the currents decayed exponentially with a single voltage-dependent time constant (63.5 s at -40 mV; 10.8 s at -130 mV). The cell input conductance decreased during the command step with the same time course as the current. On returning to the holding potential, the ensuing outward currents were accompanied by a slow increase in input conductance toward the initial values; the inward charge movement during the transient ON response (a mean of 76 nC in 8 neurons stepped from -50 to -90 mV) was completely balanced by outward charge displacement during the OFF response. The chloride movements accompanying voltage modifications were studied by estimating the chloride equilibrium potential (E(Cl)) at different holding potentials from the reversal of GABA evoked currents. [Cl(-)](i) was strongly affected by membrane potential, and at steady state it was systematically higher than expected from passive ion distribution. The transient current was blocked by substitution of isethionate for chloride and by Cl(-) channel blockers (9AC and DIDS). It proved insensitive to K(+) channel blockers, external Cd(2+), intracellular Ca(2+) chelators [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)] and reduction of [Na(+)](e). It is concluded that membrane potential shifts elicit a chloride current that reflects readjustment of [Cl(-)](i). The cell input conductance was measured over the -40/-120-mV voltage range, in control medium, and under conditions in which either the chloride or the potassium current was blocked. A mix of chloride, potassium, and leakage conductances was detected at all potentials. The leakage component was voltage independent and constant at approximately 14 nS. Conversely, gCl decreased with hyperpolarization (80 nS at -40 mV, undetectable below -110 mV), whereas gK displayed a maximum at -80 mV (55.3 nS). Thus the ratio gCl/gK continuously varied with membrane polarization (2.72 at -50 mV; 0.33 at -110 mV). These data were forced in a model of the three current components here described, which accurately simulates the behavior observed in the "resting" neuron during membrane migrations in the subthreshold potential range, thereby confirming that active K and Cl conductances contribute to the genesis of membrane potential and possibly to the control of neuronal excitability.

P-type Ca2+ current in crayfish peptidergic neurones.

Inward Ca2+ current through voltage-gated Ca2+ channels was recorded from freshly dissociated crayfish X-organ (XO) neurones using the whole-cell voltage-clamp technique. Changing the holding potential from -50 to -90 mV had little effect on the characteristics of the current-voltage relationship: neither the time course nor the amplitude of the Ca2+ current was affected. Inactivation of the Ca2+ current was observed over a small voltage range, between -35 and -10 mV, with half-inactivation at -20 mV. The activation of the Ca2+ current was modelled using Hodgkin-Huxley kinetics. The time constant of activation, &tgr; m, was 568+/-66 micros at -20 mV and decreased gradually to 171+/-23 micros at 40 mV (means +/- s.e.m., N=5). The steady-state activation, m(infinity), was fitted with a Boltzmann function, with a half-activation voltage of -7.45 mV and an apparent threshold at -40 mV. The instantaneous current-voltage relationship was adjusted using the Goldman-Hodgkin-Katz constant-field equation, giving a permeation of 4.95x10(-5 )cm s-1. The inactivation of the Ca2+ current in XO neurones was dependent on previous entry of Ca2+. Using a double-pulse protocol, the inactivation was fitted to a U-shaped curve with a maximal inactivation of 35 % at 30 mV. The time course of the recovery from inactivation was fitted with an exponential function. The time constants were 17+/-2.6 ms for a prepulse of 10 ms and 31+/-3.2 ms for a prepulse of 20 ms. The permeability sequence of the Ca2+ channels was as follows: Ba2+>Sr2+~Ca2+>>Mg2+. Other divalent cations blocked the Ca2+ current, and their effects were voltage-dependent; the potency of blockage was Cd2+~Zn2+>>Co2+~Ni2+. The peptide &ohgr; -agatoxin-IVA, a selective toxin for P-type Ca2+ channels, blocked 85 % of the Ca2+ current in XO neurones at 200 nmol l-1, but the current was insensitive to dihydropyridines, phenylalkylamines, &ohgr; -conotoxin-GVIA and &ohgr; -conotoxin-MVIIC, which are blockers of L-, N- and Q-type Ca2+ channels, respectively. From the voltage- and Ca2+-dependent kinetics, the higher permeability to Ba2+ than to Ca2+ and the higher sensitivity of the current to Cd2+ than to Ni2+, we conclude that the Ca2+ current in XO neurones is generated by high-voltage-activated (HVA) channels. Furthermore, its blockage by &ohgr; -agatoxin-IVA suggests that it is mainly generated through P-type Ca2+ channels.

Dynamics of calcium regulation of chloride currents in Xenopus oocytes.

Ca-activated Cl currents are widely expressed in many cell types and play diverse and important physiological roles. The Xenopus oocyte is a good model system for studying the regulation of these currents. We previously showed that inositol 1,4,5-trisphosphate (IP3) injection into Xenopus oocytes rapidly elicits a noninactivating outward Cl current (ICl1-S) followed several minutes later by the development of slow inward (ICl2) and transient outward (ICl1-T) Cl currents. In this paper, we investigate whether these three currents are mediated by the same or different Cl channels. Outward Cl currents were more sensitive to Ca than inward Cl currents, as shown by injection of different amounts of Ca or by Ca influx through a heterologously expressed ligand-gated Ca channel, the ionotropic glutamate receptor iGluR3. These data could be explained by two channels with different Ca affinities or one channel with a higher Ca affinity at depolarized potentials. To distinguish between these possibilities, we determined the anion selectivity of the three currents. The anion selectivity sequences for the three currents were the same (I > Br > Cl), but ICl1-S had an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, after consideration of possible errors, the hypothesis that a single type of Cl channel underlies the complex waveforms of the three different macroscopic Ca-activated Cl currents in Xenopus oocytes remains a viable alternative.

Pore mutations alter closing and opening kinetics in Shaker K+ channels.

1. We have studied the effects of mutations of amino acids in the pore (positions 447 and 449) and the elevation of extracellular [K+] on the closing and opening kinetics of Shaker B K+ channels transiently expressed in Chinese hamster ovary (CHO) cells. 2. Mutant D447E had closing and C-type inactivation kinetics which were faster than the wild-type channel. These processes were slowed by increasing extracellular [K+] and in these conditions the channels exhibited linear instantaneous current-voltage relationships. Thus, the mutation seems to produce uniform decrease of occupancy by K+ in sites along the channel pore where the cation competes with closing and C-type inactivation. 3. In other mutants also showing K+-dependent fast C-type inactivation, closing was found to be slower than in the wild-type channel and insensitive to variations in external [K+]. These characteristics were particularly apparent in mutant T449K which even in high [K+] has a non-linear instantaneous current-voltage relationship with marked saturation of the inward current recorded at negative membrane potentials. Hence, in this channel type occupation by K+ of the pore appears to be non-uniform with low occupancy of sites near the outer entrance and saturation of the sites accessible from the internal solution. 4. The results show that channel closing is influenced by changes in the pore structure leading to alterations in the occupation of the channels by permeant cations. The differential effects of pore mutations and high external [K+] on closing and C-type inactivation indicate that the respective gates are associated with separate domains of the molecule. 5. Point mutations in the pore sequence can also lead to modifications in channel opening. In general, channels with fast C-type inactivation also show a fast rising phase of activation. However, these effects appear not to be due to primary modifications of the activation process but to arise from the coupling of activation and C-type inactivation. 6. These data, demonstrating that the pore structure influences most of the gating parameters of K+ channels, give further insight into the mechanisms underlying the modulation of K+ channel function by changes in the ionic composition in the extracellular milieu.

Kinetic and pharmacological properties of the calciumm-activated chloride-current in macrovascular endothelial cells

We have studied the kinetic and pharmacological properties of the Ca 2+-activated Cl − current (I Cl,Ca) in cultured calf pulmonary artery endothelial (CPAE) cells by means of combined patch clamp and Fura-2 micro fluorescence measurements. The current was activated by loading the cells via the patch pipettes with Ca 2+-buffered solutions. Currents activated slowly at positive potentials, and decayed rapidly at negative potentials. The time constant of activation decreased at more positive membrane potentials and more elevated intracellular Ca 2+ concentrations ([Ca 2+] i). The time constant of deactivation was Ca 2+-independent and decreased at more negative potentials. Steady-state currents showed strong outward rectification, but the instantaneous current-voltage relationship was almost ohmic. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit I Cl,Ca, Half maximal block for TFP occurred at 5.7 ± 2.1 μM ( n = 16). GTPγS did not activate I Cl,Ca, but activated a Cl −- current similar to the volume-activated Cl −- current (I Cl,vol). [Ca 2+] i for half maximal activation of I Cl,Ca was voltage-dependent, and suggests that the apparent binding constant for Ca 2+ decreases with depolarization. Its value at 0 mV is 430 nM, and the binding site is 12% within the electrical field from the cytoplasmic side. The Hill-coefficient, n H, of the binding was larger than 1 and increased with depolarization. The maximal Cl − conductance at saturating [Ca 2+] i did not depend on the membrane potential. RT-PCR experiments did not provide any evidence that the endothelial Ca 2+-activated Cl − channel might be identical with a recently cloned Ca 2+-sensitive Cl − channel (CaCC).

Multiple effects of serotonin on membrane properties of trigeminal motoneurons in vitro.

Intracellular recordings from guinea pig trigeminal motoneurons (TMNs) in brain stem slices were used to determine the underlying ionic mechanisms responsible for our previously demonstrated enhancement of TMN excitability during jaw movements by serotonin (5-HT). 5-HT (0.5-100 microM) depolarized motoneurons and increased input resistance in the majority of neurons tested. Additionally, 5-HT reduced the amplitude of the postspike medium-duration afterhyperpolarization, decreased the current threshold for maintained spike discharge, and increased the maximum slope of the steady-state spike frequency-current relationship. Under voltage clamp, from holding potentials close to resting potential, 5-HT produced an inward current and a decrease in instantaneous slope conductance, suggesting a reduction in a resting K+ leak conductance (I(leak)). The instantaneous current-voltage (I-V) relationship for the inward 5-HT current (I(5-HT)) was linear throughout most of the voltage range tested. However, the steady-state I-V relationship showed some degree of inward rectification at potentials starting around -70 mV. The mean reversal potential for the instantaneous I(5-HT) was -86.2 +/- 4.5 (SE) mV (n = 9), a value slightly negative to the predicted potassium equilibrium potential of -82 mV in these neurons. In the presence of 2 mM Ba2+, 5-HT application did not produce a further reduction in input conductance, but did expose a Ba2+-insensitive residual inward current that was resistant to Cs+ application. The instantaneous I-V relationship during 5-HT application in the presence of Ba2+ was shifted downward and parallel to control, suggesting that Ba2+ and 5-HT block the same resting I(leak). The residual Ba2+- and Cs+-insensitive component of the total inward I(5-HT) was voltage independent and was blocked when the extracellular Na+ was replaced by choline, suggesting that the predominant charge carrier for this residual current is Na+. 5-HT enhanced a hyperpolarization-activated cationic current, I(h). In the presence of Ba2+, the time course of I(5-HT) resembled that of I(h) and showed a similar voltage dependence that was blocked by extracellular Cs+ (1-3 mM). The effects of 5-HT on membrane potential, input resistance, and I(h) were partially mimicked by 5-HT2 agonists and suppressed by 5-HT2 antagonists. It is concluded that 5-HT enhances TMN membrane excitability through modulation of multiple intrinsic membrane conductances. This provides for a mechanism(s) to fine tune the input-output discharge properties of these neurons, thus providing them with greater flexibility in output in response to time-varying synaptic inputs during various movements of the jaw.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line.

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to + 40 mV, with a dominating fast component and a small slower component. 3. The steady-state inactivation, h infinity, was related to potential by a Boltzmann distribution, underlying three states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by an initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O<-->I1<-->I2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m chi model. However, the best-fitted value of chi varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha m and beta m, were determined. Their functional dependence on the membrane potential was investigated.

Effect of pH on potassium and proton conductance in renal proximal tubule

The effect of intracellular and extracellular pH on potassium conductance (GK) was examined in isolated amphibian (Rana pipiens) proximal tubule cells under whole cell voltage clamp conditions. Internal perfusion of the patch pipette was used to precisely control intracellular pH. In the region of normal resting potential (-51 +/- 3 mV), raising cell pH from 6.5 to 8.0 did not significantly increase GK (1.1 +/- 0.3 vs. 1.3 +/- 0.3 nS; P > 0.08, n = 8). Similar elevations in external (bath) pH had even less of an effect on GK. In contrast, when cells were voltage clamped to 30 mV more negative than the resting potential, raising internal pH from 6.5 to 8.0 did increase GK from 1.05 +/- 0.3 to 1.8 +/- 0.5 nS (P < 0.04; n = 8). These results suggest that modest changes in pH have little effect on GK, except at large negative potentials. In the process of examining the pH dependence of GK, a slowly activating, voltage-dependent conductance of 7.5 +/- 1 nS (n = 20; for 20 microns cells) was observed during cell depolarization. Although the instantaneous current-voltage relation of this conductance was linear, its marked voltage dependence produced an apparent steady-state rectification, with Gm = 0.5 +/- 0.2 nS and Gout = 9.0 +/- 1 nS (n = 11). Outward current was reversibly blocked by 3 mM Cu, Cd, or Co. In the absence of Na, K, and Ca (and only trace amounts of Cl), rapid changes in bath pH from 6.5 to 8.0 shifted the steady-state reversal potential (Erev) by -37 +/- 4 mV (n = 9) and the instantaneous Erev by -56 +/- 4 mV (n = 9). These shifts in Erev were consistent with a hydrogen ion conductance (GH), similar to what has been reported for snail neuron, neutrophils, alveolar epithelial cells, and phagocytes. Since the magnitude of this GH would be insignificant at resting cell pH and membrane potential, its role in renal proximal tubule under normal conditions is somewhat obscure. Nonetheless, in pathological situations, GH could function to prevent acid overload during any process that depolarizes the cell, such as low temperature or metabolic inhibition.