Steady-state Current-voltage Relationship Research Articles

Molnupiravir, a ribonucleoside antiviral prodrug against SARS-CoV-2, alters the voltage-gated sodium current and causes adverse events

Molnupiravir (MOL) is a ribonucleoside prodrug for oral treatment of COVID-19. Common adverse effects of MOL are headache, diarrhea, and nausea, which may be associated with altered sodium channel function. Here, we investigated the effect of MOL on voltage-gated Na+ current (INa) in pituitary GH3 cells. We show that MOL had distinct effects on transient and late INa, in combination with decreased time constant in the slow component of INa inactivation. The 50% inhibitory concentration (IC50) values of MOL for suppressing transient and late INa were 26.1 and 6.3 μM, respectively. The overall steady-state current-voltage relationship of INa remained unchanged upon MOL exposure. MOL-induced alteration of INa may lead to changes in physiological function through sodium channels. Apart from its effect on inhibiting RNA virus replication, MOL exerts inhibitory effects on plasmalemma INa, which might constitute an additional yet crucial underlying mechanism of its pharmacological activity or adverse events.

Non-trivial dynamics in a model of glial membrane voltage driven by open potassium pores

Despite the molecular evidence that a nearly linear steady-state current-voltage relationship in mammalian astrocytes reflects a total current resulting from more than one differentially regulated K+ conductance, detailed ordinary differential equation (ODE) models of membrane voltage Vm are still lacking. Various experimental results reporting altered rectification of the major Kir currents in glia, dominated by Kir4.1, have motivated us to develop a detailed model of Vm dynamics incorporating the weaker potassium K2P-TREK1 current in addition to Kir4.1, and study the stability of the resting state Vr. The main question is whether, with the loss of monotonicity in glial I-V curve resulting from altered Kir rectification, the nominal resting state Vr remains stable, and the cell retains the trivial, potassium electrode behavior with Vm after EK. The minimal two-dimensional model of Vm near Vr showed that an N-shape deformed Kir I-V curve induces multistability of Vm in a model that incorporates K2P activation kinetics, and nonspecific K+ leak currents. More specifically, an asymmetrical, nonlinear decrease of outward Kir4.1 conductance, turning the channels into inward rectifiers, introduces instability of Vr. That happens through a robust bifurcation giving birth to a second, more depolarized stable resting state Vdr > −10 mV. Realistic recordings from electrographic seizures were used to perturb the model. Simulations of the model perturbed by constant current through gap junctions and seizure-like discharges as local field potentials led to depolarization and switching of Vm between the two stable states, in a downstate-upstate manner. In the event of prolonged depolarizations near Vdr, such catastrophic instability would affect all aspects of the glial function, from metabolic support to membrane transport, and practically all neuromodulatory roles assigned to glia.

Apost Gurney quantum mechanical perspective on the electrolysis of water: ion neutralization in solution.

Electron fluxes crossing the interface between a metallic conductor and an aqueous environment are important in many fields; hydrogen production, environmental scanning tunnelling microscopy, scanning electrochemical microscopy being some of them. Gurney (Gurney 1931 Proc. R. Soc. Lond.134, 137 (doi:10.1098/rspa.1931.0187)) provided in 1931 a scheme for tunnelling during electrolysis and outlined conditions for it to occur. We measure the low-voltage current flows between gold electrodes in pure water and use the time-dependent behaviour at voltage switch-on and switch-off to evaluate the relative contribution to the steady current arising from tunnelling of electrons between the electrodes and ions in solution and from the neutralization of ions adsorbed onto the electrode surface. We ascribe the larger current contribution to quantum tunnelling of electrons to and from ions in solution near the electrodes. We refine Gurney's barrier scheme to include solvated electron states and quantify energy differences using updated information. We show that Gurney's conditions would prevent the current flow at low voltages we observe but outline how the ideas of Marcus (Marcus 1956 J. Chem. Phys.24, 966-978 (doi:10.1063/1.1742723)) concerning solvation fluctuations enable the condition to be relaxed. We derive an average barrier tunnelling model and a multiple pathways tunnelling model and compare predictions with measurements of the steady-state current-voltage relation. The tunnelling barrier was found to be wide and low in agreement with other experimental studies. Applications as a biosensing mechanism are discussed that exploit the fast tunnelling pathways along molecules in solution.

A single Markov-type kinetic model reliably accounts for the macroscopic currents of all human voltage-gated sodium channel isoforms

A single Markov-type kinetic model reliably accounts for the macroscopic currents of all human voltage-gated sodium channel isoforms

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl−/1HCO3− exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.

Remote Modulation of Current Transfer from Dendritic Glutamatergic Synapses by GABA-ergic Synapses of the Somatic Zone of Motoneurons: a Simulation Study

Until now, information concerning spatial interaction of postsynaptic excitation and inhibition in neuronal dendrites remains rather limited. In model experiments, we studied spatial effects of tonic co-activation of GABA-ergic synapses situated on the soma and axon hillock of a motoneuron and dendritic glutamatergic synapses with receptors sensitive or insensitive to N-methyl-D-aspartate. We analyzed distribution maps of the transmembrane potentials and excitatory currents transferred toward the soma over the reconstructed dendritic arborization of a rat abducens motoneuron (three-dimensional reconstruction). In the motoneuron, isolated tonic excitation of glutamatergic synapses induced two stable states of low (downstate) or high (upstate) spatially heterogeneous dendritic depolarization, which decayed with unequal rates along different dendritic paths. In this case, the local steady-state current-voltage relation of the dendritic membrane became N-shaped due to a limb of the negative slope within a certain voltage range. The upstate corresponding to plateau potentials associated with stereotyped motor activity patterns was analyzed in detail. In this state, most proximal dendritic sites were the main sources of the excitatory current reaching the soma, while the contribution from distal sites was negligible. Co-activation of GABA-synapses located at the soma and axon hillock reduced this depolarization and shifted the main excitatory current source from a perisomatic location to the middle, structurally more complex, region of the dendritic arborization. The more remote dendritic region having a greater membrane area and receiving a greater number of synaptic contacts became directly involved in the supply of the trigger zone by the excitatory current. We suggest that a special, not described earlier, operational mechanism of postsynaptic inhibition is manifested in the above spatial effects of activation of strategically located inhibitory synapses, and that the list of known crucial inhibitory mechanisms (namely hyperpolarization and shunting of the postsynaptic membrane) must be expanded.

Ion Channel Density Regulates Switches between Regular and Fast Spiking in Soma but Not in Axons

The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking) shows a continuous relationship between frequency and stimulation current (f-Istim) and, thus, an arbitrarily low frequency at threshold current; Type 2 (fast spiking) shows a discontinuous f-Istim relationship and a minimum threshold frequency. In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship (a prerequisite for Type 1 dynamics and associated with this type of dynamics in the hippocampal model). In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane.

Potassium Channel Silencing by Constitutive Endocytosis and Intracellular Sequestration

Tandem of P domains in a weak inwardly rectifying K(+) channel 1 (TWIK1) is a K(+) channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT(1)R or adrenoreceptor alpha2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G(i) protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.

5-HT 1A receptor-mediated activation of a G-protein-coupled inwardly rectifying K + current in rat medial preoptic area neurons

The medial preoptic area plays an important role in the regulation of sexual behavior, and serotonin (5-hydroxytryptamine, 5-HT) exerts an inhibitory effect on sexual behavior by acting on the medial preoptic area region. This study was designed to clarify the inhibitory effect of 5-HT on the medial preoptic area neurons and to elucidate the electrophysiological mechanisms involved in the action of 5-HT. Superfusion of 100 nM 5-HT hyperpolarized the membrane potential and inhibited the action potential firing. When the membrane potential was stepped to various potentials, the inward K + currents were potentiated in the presence of 100 nM 5-HT. When the concentration of K + in the external solution was increased from 5 mM to 30 mM, 5-HT markedly potentiated the inward K + currents. In the steady-state current–voltage relationship, the 5-HT-activated inward current was carried by K + ions and showed characteristics typical of an inwardly rectifying K + current. The 5-HT-activated K + current was mimicked by a 5-HT 1A receptor agonist, (±)-8-hydroxy-2-dipropylaminotetralin hydrobromide, and was reversibly blocked by a 5-HT 1A receptor antagonist, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide, but not by a 5-HT 2 receptor antagonist, ketanserin. The 5-HT-activated K + current was sensitively blocked by Ba 2+, but not by 4-aminopyridine, and was completely suppressed by N-ethylmaleimide. These results indicate that 5-HT-induced hyperpolarization of the medial preoptic area neurons occurs as a result of activation of the G-protein-coupled inwardly rectifying K + currents by 5-HT 1A receptors.

Mutation of KCNE3 alters activation of KCNQ1 but not channel block by Ba 2+

In secretory epithelial cells, KCNQ1 associates with the KCNE3 subunit to form a constitutively active, voltage-insensitive channel. We demonstrate by RT-PCR the presence of KCNQ1 and KCNE3 mRNAs in the human liver secretory cell line, Mz-ChA1, and show using immunofluorescence that KCNQ1 is expressed in rat liver secretory ducts. In the heart KCNQ1 interacts with the KCNE1 subunit to form a voltage-gated K+ channel. To determine mechanisms of subunit interaction, we expressed KCNQ1 in Xenopus oocytes and determined the effects of the blocker Ba2+ on current-voltage relationships. Bath Ba2+ (5 mM) slows activation and reduces (24%) KCNQ1 currents. Ba2+ causes the constitutively active K+ currents of KCNQ1 co-expressed with KCNE3 to become slowly-activating and dramatically reduced (42%). KCNQ1+KCNE1 currents are very slowly activated and Ba2+ has little (16%) effect. The dose-inhibition curve for Ba2+ on KCNQ1 shows two binding components with affinities at 10 μM and 20 mM. The curve for KCNQ1+KCNE1 shows only one component (20 μM) and KCNQ1+KCNE3 shows one component (100 μM). We also determined the effects of a V72T mutation in KCNE3. KCNQ1+KCNE3(V72T) currents are slowly-activating, like KCNQ1+KCNE1, while near steady-state current-voltage relationships are similar to KCNQ1 alone and inhibition by Ba2+ (2 mM) is identical to that of KCNQ1+KCNE3. These results suggest that the site of regulation of KCNQ1 by KCNE1 or KCNE3 involves both the voltage-sensitive gating mechanism and extracellular pore region of KCNQ1. (NIH 62465)

Generation of Very Slow Neuronal Rhythms and Chaos Near the Hopf Bifurcation in Single Neuron Models

We have presented a new generation mechanism of slow spiking or repetitive discharges with extraordinarily long inter-spike intervals using the modified Hodgkin-Huxley equations (Doi and Kumagai, 2001). This generation process of slow firing is completely different from that of the well-known potassium A-current in that the steady-state current-voltage relation of the neuronal model is monotonic rather than the N-shaped one of the A-current. In this paper, we extend the previous results and show that the very slow spiking generically appears in both the three-dimensional Hodgkin-Huxley equations and the three dimensional Bonhoeffer-van der Pol (or FitzHugh-Nagumo) equations. The generation of repetitive discharges or the destabilization of the unique equilibrium point (resting potential) is a simple Hopf bifurcation. We also show that the generation of slow spiking does not depend on the stability of the Hopf bifurcation: supercritical or subcritical. The dynamics of slow spiking is investigated in detail and we demonstrate that the phenomenology of slow spiking can be categorized into two types according to the type of the corresponding bifurcation of a fast subsystem: Hopf or saddle-node bifurcation.

Apparent charge of binding site in ion-translocating enzymes: kinetic impact

Recently, we presented a general scope for the nonlinear electrical properties of enzymes E which catalyze translocation of a substrate S with charge number z(S) through lipid membranes (Boyd et al. J. Membr. Biol. 195:1-12, 2003). In this study, the voltage sensitivity of the enzymatic reaction cycle has been assigned to one predominant reversible reaction step, i.e. the reorientation of either E or ES in the electric field, leaving the reorientation of the alternate state (ES or E) electroneutral, respectively. With this simplification, the steady-state current-voltage relationships (IV) assumed saturation kinetics like in Michaelis-Menten systems. Here, we introduce an apparent charge number z(E) of the unoccupied binding site of the enzyme, which accounts for the impact of all charged residues in the vicinity of the physical binding site. With this more realistic concept, the occupied binding site assumes an apparent charge of z(ES) = z(E) + z(S), and IV does not saturate any more in general, but exponentially approaches infinite or zero current for large voltage displacements from equilibrium. These nonlinear characteristics are presented here explicitly. They are qualitatively explained in a mechanistic way, and are illustrated by simple examples. We also demonstrate that the correct determination of the model parameters from experimental data is still possible after incorporating z(E) and its corollaries into the previous model of enzyme-mediated ion translocation.

Effects of norfluoxetine on the action potential and transmembrane ion currents in canine ventricular cardiomyocytes.

Norfluoxetine is the most important active metabolite of the widely used antidepressant compound fluoxetine. Although the cellular electrophysiological actions of fluoxetine are well characterized in cardiac cells, little is known about the effects of its metabolite. In this study, therefore, the effects of norfluoxetine on action potential (AP) configuration and transmembrane ion currents were studied in isolated canine cardiomyocytes using the whole cell configuration of patch clamp techniques. Micromolar concentrations of norfluoxetine (1-10 microM) modified AP configuration: amplitude and duration of the AP and maximum velocity of depolarization were decreased in addition to depression of the plateau and elimination of the incisura of AP. Voltage clamp experiments revealed a concentration-dependent suppression of both L-type Ca(2+) current, I(Ca) (EC(50)=1.13+/-0.08 microM) and transient outward K(+) current, I(to) (EC(50)=1.19+/-0.17 microM) having Hill coefficients close to unity. The midpoint potential of the steady-state inactivation of I(Ca) was shifted from -20.9+/-0.75 mV to -27.7+/-1.35 mV by 3 microM norfluoxetine ( P<0.05, n=7). No such shift in the steady-state inactivation curve was observed in the case of I(to). Similarly, norfluoxetine caused no change in the steady-state current-voltage relationship of the membrane or in the density of the inward rectifier K(+) current, I(K1). All these effects of norfluoxetine developed rapidly and were fully reversible. Comparing present results with those obtained previously with fluoxetine, it can be concluded that norfluoxetine displays stronger suppression of cardiac ion channels than fluoxetine. Consequently, the majority of the cardiac side effects observed during fluoxetine treatment are likely to be attributed to its metabolite norfluoxetine.

Identification of a Heteromeric Interaction That Influences the Rectification, Gating, and pH Sensitivity of Kir4.1/Kir5.1 Potassium Channels

Heteromultimerization between different potassium channel subunits can generate channels with novel functional properties and thus contributes to the rich functional diversity of this gene family. The inwardly rectifying potassium channel subunit Kir5.1 exhibits highly selective heteromultimerization with Kir4.1 to generate heteromeric Kir4.1/Kir5.1 channels with unique rectification and kinetic properties. These novel channels are also inhibited by intracellular pH within the physiological range and are thought to play a key role in linking K+ and H+ homeostasis by the kidney. However, the mechanisms that control heteromeric K+ channel assembly and the structural elements that generate their unique functional properties are poorly understood. In this study we identify residues at an intersubunit interface between the cytoplasmic domains of Kir5.1 and Kir4.1 that influence the novel rectification and gating properties of heteromeric Kir4.1/Kir5.1 channels and that also contribute to their pH sensitivity. Furthermore, this interaction presents a structural mechanism for the functional coupling of these properties and explains how specific heteromeric interactions can contribute to the novel functional properties observed in heteromeric Kir channels. The highly conserved nature of this structural association between Kir subunits also has implications for understanding the general mechanisms of Kir channel gating and their regulation by intracellular pH.

The inhibitory effects of dantrolene on action potential-induced calcium transients in cultured rat dorsal root ganglion neurons

We investigated the actions of dantrolene Ca2+-induced on Ca2+-release (CICR) evoked by action potentials in cultured rat sensory neurons. The effect of dantrolene on action potential after-depolarization and voltage-activated calcium currents was studied in cultured neonatal rat dorsal root ganglion cells (DRG) using the whole-cell patch-clamp technique. Depolarizing current injection evoked action potentials and depolarizing after-potentials, which are activated as a result of CICR following a single action potential in some cells. The type of after-potentials was determined by inducing action potentials from the resting membrane potential. Extracellular application of dantrolene (10 mM) abolished after-depolarizations without affecting action potential properties. Furthermore, dantrolene significantly reduced repetitive action potentials after depolarizing current injection into these neurons, but had no significant effect on the steady-state current voltage relationship of calcium currents in these neurons. We conclude that dantrolene inhibits the induction of action potential after depolarizations by inhibiting CICR in cultured rat sensory neurons.

Voltage dependence of the apparent affinity for external Na(+) of the backward-running sodium pump.

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.

ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart.

The full-length cDNAs of two different alpha-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different beta-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3-15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 +/- 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 microM 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 microM), diazoxide (300 microM) and metabolic inhibition increased the slope conductance measured at -55 mV by a factor of 9.0 (+/-1.8), 2.5 (+/-0.2) and 3.9 (+/-1.7), respectively. The effects were reversed by glibenclamide (1 microM). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage.

Ca(2+)-activated Cl(-) currents (I(Cl,Ca)) were examined using fluorescence confocal microscopy to monitor intracellular Ca(2+) liberation evoked by flash photolysis of caged inositol 1,4, 5-trisphosphate (InsP(3)) in voltage-clamped Xenopus oocytes. Currents at +40 mV exhibited a steep dependence on InsP(3) concentration ([InsP(3)]), whereas currents at -140 mV exhibited a higher threshold and more graded relationship with [InsP(3)]. Ca(2+) levels required to half-maximally activate I(Cl,Ca) were about 50% larger at -140 mV than at +40 mV, and currents evoked by small Ca(2+) elevations were reduced >25-fold. The half-decay time of Ca(2+) signals shortened at increasingly positive potentials, whereas the decay of I(Cl,Ca) lengthened. The steady-state current-voltage (I-V) relationship for I(Cl,Ca) exhibited outward rectification with weak photolysis flashes but became more linear with stronger stimuli. Instantaneous I-V relationships were linear with both strong and weak stimuli. Current relaxations following voltage steps during activation of I(Cl,Ca) decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at -160 mV. We conclude that InsP(3)-mediated Ca(2+) liberation activates a single population of Cl(-) channels, which exhibit voltage-dependent Ca(2+) activation and voltage-independent instantaneous conductance.

Ca2+ and cAMP-activated Cl- conductances mediate Cl- secretion in a mouse renal inner medullary collecting duct cell line.

1. The nature of Cl- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated. 2. Extracellular ATP (100 microM) stimulated a transient increase in both whole-cell Cl- conductance and intracellular free Ca2+. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl- conductance. Pre-loading cells with the Ca2+ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin. 3. The Ca2+-activated whole-cell Cl- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation. 4. Ca2+-activated and forskolin-activated Cl- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl-/HCO3- but independent of the presence of apical bathing medium Na+ and Cl-/HCO3-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+-activated Cl- channel (mCLCA1) mRNA in mIMCD-K2 cells. 7. Ca2+ and forskolin-activated Cl- conductances participate in anion secretion by IMCD.

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals.

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.