Transinhibition and voltage-gating in a fungal nitrate transporter.

We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. (Zhou, J.J., Trueman, L.J., Boorer, K.J., Theodoulou, F.L., Forde, B.G., Miller, A.J. 2000. A high-affinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894-9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H+-NO3- symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I(NO3-). We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H+ + NO3-)o, the product concentration inside, [P] = [H+]i(2) x [NO3-]i, may rise substantially near the plasma membrane, violating the condition [P] << [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K(1/2) approximately 20 microM for I(NO3-) at pHo from Zhou et al. is confirmed. According to our analysis, NrtA operates between about 0.2 and 0.6 of the electrical distance in the membrane (outside 0, inside 1). In absence of thermodynamic gradients, the predominant orientation of the binding site(s) is probably inwards. The activity of the enzyme is sensitive to the transmembrane voltage, V, with an apparent gating charge of +1.0 +/- 0.5 for inactivation, and transition probabilities of 0.3-1.3 s(-1) at V = 0. This gating mode impedes loss of cellular NO3- during depolarization.

Modeling light-induced currents in the eye of Chlamydomonas reinhardtii.

Rhodopsin-mediated electrical events in green algae have been recorded in the past from the eyes of numerous micro-algae like Haematococcus pluvialis, Chlamydomonas reinhardtii and Volvox carteri. However, the electrical data gathered by suction-pipette techniques could be interpreted in qualitative terms only. Here we present two models that allow a quantitative analysis of such results: First, an electrical analog circuit for the cell in suction pipette configuration is established. Applying this model to experimental data from unilluminated cells of C. reinhardtii yields a membrane conductance of about 3 Sm(-2). Furthermore, an analog circuit allows the determination of the photocurrent fraction that is recorded under experimental conditions. Second, a reaction scheme of a rhodopsin-type photocycle with an early Ca(2+) conductance and a later H(+) conductance is presented. The combination of both models provides good fits to light-induced currents recorded from C. reinhardtii. Finally, it allowed the calculation of the impact of each model parameter on the time courses of observable photocurrent and of inferred transmembrane voltage. The reduction of the flash-to-peak times at increasing light intensities are explained by superposition of two kinetically distinct rhodopsins and by assuming that the Ca(2+)-conducting state decays faster at more positive membrane voltages.

Impact of apoplast volume on ionic relations in plant cells.

Ionic relations of plant cells with free-running transmembrane voltage, V, can be modelled by electrocoupling of known, V-relevant and V-gated transporters. For cells in vitro, with constant external substrate concentrations, alternating states of solute uptake and release can be predicted. Here the model is extended to treat cells with limited external substrates, e.g., parenchyma cells in situ with a small external space, the apoplast. This model accounts also for cytoplasmic H+ buffering and apoplastic buffering of H+, K+ and Ca2+ by fixed anions. In general, the model converges to thermodynamic equilibrium for K+ between apoplast and symplast, and to equal steady-state rates of uptake and release for Cl- and H+. Oscillations of this model are rare and very sensitive to the volume portion of the apoplast.

Effects of Na(+) on the predominant K(+) channel in the tonoplast of Chara: decrease of conductance by blocks in 100 nanosecond range and induction of oligo- or poly-subconductance gating modes.

We present three mechanisms by which Na(+) inhibits the open channel currents of the predominant K(+) channel in the tonoplast of Chara corallina: (i) Fast block, i.e., short (100 ns range) interruptions of the open channel current which are determined by open channel noise analysis, (ii): Oligo-subconductance mode, i.e., a gating mode which occurs preferentially in the presence of Na(+); this mode comprises a discrete number (here 3) of open states with smaller conductances than normal, and (iii): Polysubconductance mode, i.e., a gating mode with a nondiscrete, large number (>30) of states with smaller conductances than the main open channel conductance. This novel mode has also been observed only in the presence of Na(+).

Three types of membrane excitations in the marine diatom Coscinodiscus wailesii.

Three types of electrical excitation have been investigated in the marine diatom Coscinodiscus wailesii. I: Depolarization-triggered, transient Cl(-) conductance, G(Cl)(t), followed by a transient, voltage-gated K(+) conductance, G(K), with an active state a and two inactive states i(1) and i(2) in series (a-i(1)-i(2)). II: Similar G(Cl)(t) as in Type-I but triggered by hyperpolarization; a subsequent increase of G(K) in this type is indicated but not analyzed in detail. III: Hyperpolarization-induced transient of a voltage-gated activity of an electrogenic pump (i(2)-a-i(2)), followed by G(Cl)(t) as in Type-II excitations. Type-III with pump gating is novel as such. G(Cl)(t) in all types seems to reflect the mechanism of InsP(-)(3) and Ca(2+)-mediated G(Cl)(t) in the action potential in Chara (Biskup et al., 1999). The nonlinear current-voltage-time relationships of Type-I and Type-III excitations have been recorded under voltage-clamp using single saw-tooth command voltages (voltage range: -200 to +50 mV, typical slope: +/-1 Vs(-1)). Fits of the corresponding models to the experimental data provided numerical values of the model parameters. The statistical significance of these solutions is investigated. We suggest that the original function of electrical excitability of biological membranes is related to osmoregulation which has persisted through evolution in plants, whereas the familiar and osmotically neutral action potentials in animals have evolved later towards the novel function of rapid transmission of information over long distances.

Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel.

Effects of threonine substitution by glutamine at position 256 in the pore of the KAT1 channel have been investigated by voltage-clamp, using heterologous gene expression in Xenopus oocytes. The major discrepancy in T256Q from the wild-type channel (wt) was cation specific. While K(+) currents were reduced in a largely scalar fashion, the NH(4)(+) current exhibited slow, voltage-dependent inhibition during hyperpolarization. The same effects could be induced in wt, or intensified in T256Q, by addition of the impermeant cation methylammonium (MA(+)) to the bath. This stresses that both the mutation and MA(+) affect a mechanism already present in the wt. Assuming that current inhibition could be described as entry of the channel into an inactive state, we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme, where C (closed) and I (inactivated) are nonconductive states, and O is an open state allowing K(+) and NH(4)(+) passage. The key reaction is the transition I-O. This cation-sensitive transition step ensures release of the channel from the inactive state and is approximately 30 times smaller in T256Q compared to wt. It can be inhibited by external MA(+) and is stimulated strongly by K(+) and weakly by NH(4)(+). This sensitivity of gating to external cations may prevent K(+) leakage from cation-starved cells.

Electrophysiology of the marine diatom Coscinodiscus wailesii IV: types of non-linear current-voltage-time relationships recorded with single saw-tooth voltage-clamp experiments.

Electrophysiological states of the marine diatom Coscinodiscus wailesii are known to change spontaneously in the temporal range of seconds. In order to assess the genuine current-voltage-time relationships of individual states in less than a second, voltage-clamp experiments have been carried out using single sweeps of saw-tooth shaped command voltages. This method is introduced with model calculations. Plotting the results in current-voltage coordinates provides convenient access to several electrophysiological entities, such as absence of drift (smoothly closed IV loops), membrane capacitance (by I jump at sign reversal of dV/dt), and ohmic conductances (in linear regions of the current-voltage relationship), as well as equilibrium voltage (internal intersection of capacitance-corrected, 8-shaped tracings) and coarse gating kinetics (rise or fall of capacitance-corrected I at sign reversal of dV/dt) of a voltage-sensitive ion conductance. From electrophysiological measurements with double-barreled glass-microelectrodes on C. wailesii, several distinct types of current-voltage loops are presented. Most of the data, including recordings from electrical excitation, can be interpreted as temporal relaxations of voltage-sensitive conductances for K(+) and Cl(-). A more detailed analysis of the effect of tetraethylammonium (TEA(+)) shows that 10 and 20 mM TEA(+) inhibit the K(+) conductance in C. wailesii only by up to about 20% but predominantly via a K(+) outward rectifier.

Calcium release from InsP3-sensitive internal stores initiates action potential in Chara.

Neomycin and U73122 are known to suppress inositol 1,4,5-trisphosphate (InsP3) production by inhibition of phospholipase C. We studied the effects of these inhibitors on the excitatory currents, Iex, in Chara corallina under voltage-clamp conditions. Computer simulations of the experimental effects by a minimum model for the excitatory reaction pathway allow the assignment of the inhibitory effects to one specific reaction step, i.e. the release of Ca2+ from InsP3-sensitive internal stores. In contrast, the inhibitory effect of La3+ on Iex suggests inactivation of Cl- channels. Furthermore, ryanodine-sensitive Ca2+ stores seem to be irrelevant for electrical excitation in Chara.

Electrocoupling of ion transporters in plants: interaction with internal ion concentrations.

There are five major electroenzymes in the plasmalemma of plant cells: a driving electrogenic pump, inward and outward rectifying K+ channels, a Cl--2H+ symporter, and Cl--channels. It has been demonstrated previously (Gradmann, Blatt & Thiel 1993, J. Membrane Biol. 136:327-332) how voltage-gating of these electroenzymes causes oscillations of the transmembrane voltage (V) at constant substrate concentrations. The purpose of this study is to examine the interaction of the same transporter ensemble with cytoplasmic concentrations of K+ and Cl-. The former model system has been extended to account for changing internal concentrations. Constant-field theory has been applied to describe the influence of ion concentrations on current-voltage relationships of the active channels. The extended model is investigated using a reference set of model parameters. In this configuration, the system converges to stable slow oscillations with intrinsic changes in cytoplasmic K+ and Cl- concentrations. These slow oscillations reflect alternation between a state of salt uptake at steady negative values of V and a state of net salt loss at rapidly oscillating V, the latter being analogous to the previously reported oscillations. By switching off either concentration changes or gating, it is demonstrated that the fast oscillations are mostly due to the gating properties of the Cl- channel, whereas the slow oscillations are controlled by the effect of the Cl- concentration on the current. The sensitivity of output results y (e.g., frequency of oscillations) to changes of the model parameters x (e.g., maximum Cl- conductance) has been investigated for the reference system. Further examples are presented where some larger changes of specific model parameters cause fundamentally different behavior, e.g., convergence towards a stable state of only the fast oscillations without intrinsic concentration changes, or to a steady-state without any oscillations. The main and general result of this study is that the osmotic status of a plant cell is stabilized by the ensemble of familiar electroenzymes through oscillatory interactions with the internal concentrations of the most abundant ions. This convergent behavior of the stand-alone system is an important prerequisite for osmotic regulation by means of other physiological mechanisms, like second messengers and gating modifiers.

Kinetics of high-affinity K+ uptake in plants, derived from K(+)-induced changes in current-voltage relationships. A modelling approach to the analysis of carrier-mediated transport.

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K(+)-H+ symporter, responsible for high-affinity K+ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K+ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K(+)-H+ symport-mediated current with a parallel, outward non-linear K+ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K+ concentration and the cytoplasmic K+ concentration. Symport activity is a complex function of the external K+ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K+ is in the millimolar range and the membrane depolarises. High cytoplasmic K+ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K+ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed.

Kinetic analysis of Ca2+/K+ selectivity of an ion channel by single-binding-site models.

Current-voltage relationships of a cation channel in the tonoplast of Beta vulgaris, as recorded in solutions with different activities of Ca2+ and K+ (from Johannes & Sanders 1995, J. Membrane Biol. 146:211-224), have been reevaluated for Ca2+/K+ selectivity. Since conversion of reversal voltages to permeability ratios by constant field equations is expected to fail because different ions do not move independently through a channel, the data have been analyzed with kinetic channel models instead. Since recent structural information on K+ channels show one short and predominant constriction, selectivity models with only one binding site are assumed here to reflect this region kinetically. The rigid-pore model with a main binding site between two energy barriers (nine free parameters) had intrinsic problems to describe the observed current-saturation at large (negative) voltages. The alternative, dynamic-pore model uses a selectivity filter in which the binding site alternates its orientation (empty, or occupied by either Ca2+ or K+) between the cytoplasmic side and the luminal side within a fraction of the electrical distance and in a rate-limiting fashion. Fits with this model describe the data well. The fits yield about a 10% electrical distance of the selectivity filter, located about 5% more cytoplasmic than the electrical center. For K+ translocation, reorientation of the unoccupied binding site (with a preference of about 6:5 to face the lumenal side) is rate limiting. For Ca2+, the results show high affinity to the binding site and low translocation rates (<1% of the K+ translocation rate). With the fitted model Ca2+ entry through the open channel has been calculated for physiological conditions. The model predicts a unitary open channel current of about 100 fA which is insensitive to cytoplasmic Ca2+ concentrations (between 0.1 and 1 microM) and which shows little sensitivity to the voltage across the tonoplast.

K(+)-sensitive gating of the K+ outward rectifier in Vicia guard cells.

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1-30 mM K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of -200 mV, clamp steps to voltages between -150 and +50 mV in 0.1 mM K+ activated current through outward-rectifying K+ channels (IK,out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]o shifted the voltage-sensitivity of IK,out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]o was evident in the kinetics of IK,out activation and deactivation, as well as the steady-state conductance-(g kappa-) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near -130 mV in 0.1 mM K+, -80 mV in 1.0 mM K+, and -20 mV in 10 mM K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+ > or = Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mM was without effect on the voltage-dependence of g kappa or on IK,out activation kinetics, although 10 mM [Ca2+]o accelerated current deactivation at voltages negative of -75 mV. At any one voltage, increasing [K+]o suppressed g kappa completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mM with clamp voltages near -100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of IK,out gating; the results implicate K(+)-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K+ ions outside affects the distribution between closed states of the channel.

Oscillatory interactions between voltage gated electroenzymes.

The activities of the major ion pathways in the plasma membranes of plants are sensitive to the membrane voltage, V. Therefore, these 'electroenzymes' interact with each other via the free running voltage under physiological conditions. A physical background is given here, of how to calculate these interactions on the basis of experimental data on these electroenzymes. Simplifying model calculations with five major electroenzymes from plant cells (H(+) pump, inward and outward rectifying channels for K(+), a Cl(-) channel, and a 2H(+)/Cl(-) symporter) show that osmotic relations are balanced in the long-term not by an appropriate steady-state, but by alternation between a state of salt uptake at V < < E(K) (the Nernst equilibrium voltage for K(+) diffusion) and a state of salt loss at V > E(K). Several specific properties of the model are discussed numerically, e.g. minimum configuration for oscillations (with two electroenzymes), temperature-compensation, the physiological impact of fast gating in plant membranes, and solution of possible paradoxes, such as flux stimulation by conductance inhibition.

Reaction kinetics of the vacuolar H(+)-pumping ATPase in beta vulgaris.

Vacuolar-type H(+)-ATPases (V-ATPases) are ubiquitous in eukaryote endomembranes, where they are responsible for lumenal acidification. The ratios of H+ translocated per ATP hydrolyzed (which may be important in controlling the activity of these pumps) have previously been found to be variable, noninteger and sensitive to cytosolic as well as lumenal pH. The mechanistic implications of these findings are explored here with reaction kinetics. Experimental data for this analysis comprise supra- and superlinear V-ATPase current-voltage relationships, isolated as bafilomycin-sensitive currents in vacuolar membranes from Beta using the "whole vacuole" patch clamp configuration. Whereas simple models with one reaction cycle fail to provide an adequate description of the data (mainly because of a weak sensitivity of the zero-current voltage to the transmembrane pH gradient), a model with two linked reaction loops allowing partial coupling of H+ translocation to ATP hydrolysis does provide good descriptions. All experimental data obtained with the same vacuolar pH (4.3) could be reduced to a model with eleven independent parameters. Best fits have been obtained on the basis of a binding domain possessing 3 H+ binding sites per ATP hydrolyzed and a net charge of -2 when unoccupied. The enzyme could reorientate its access site between the vacuolar and cytoplasmic side when zero, one or three H+ are bound.

Small inward rectifying K+ channels in coleoptiles: inhibition by external Ca2+ and function in cell elongation.

Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca(2+)-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca(2+)-binding and -release, respectively (rate constants >> 10(3) sec-1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10(3) sec-1). With 10 mM K+ and 1 mM Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of -105 +/- 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mM Ca2+) and for conductance (1.2 mM Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.

Primary structure of the plasma membrane H(+)-ATPase from the halotolerant alga Dunaliella bioculata.

P-type ATPase-specific oligodeoxyribonucleotides were used to obtain a fragment of the H(+)-ATPase of the salt tolerant alga Dunaliella bioculata by polymerase chain reaction (PCR). This fragment served as a probe in screening a cDNA-library from this organism. The complete primary structure of the ATPase protein (DBPMA1) was deduced from sequencing a 4.7 kb cDNA clone. The protein shows highest homology to H(+)-ATPases from higher plants and fungi (43% identity, 67% similarity) but has a higher calculated molecular mass (123 kDa). The latter can be assigned mainly to an additional hydrophilic domain between transmembrane segments VI and VII and to an extended carboxyterminus. These unusual structural features of DBPMA1 are interpreted in terms of providing regulatory sites of the enzyme. Southern blot analysis suggests the presence of only a single copy of the gene in the haploid D. bioculata genome. To investigate the role of the H(+)-ATPase in the adaption of D. bioculata to different external NaCl concentrations, we employed northern blot analyses. The results indicate that the pma1 transcript level of cells growing in salinities between 0.1 and 3 M NaCl is not directly correlated with the external salt concentration.

Na+ transport in Acetabularia bypasses conductance of plasmalemma.

Na(+)-selective microelectrodes with the sensor ETH 227 have been used to measure the cytoplasmic Na+ concentration, [Na+]c, in Acetabularia. In the steady-state, [Na+]c is about 60 mM (external 460 mM). Steps in external Na+ concentration, [Na+]o, cause biexponential relaxations of [Na+]c which have formally been described by a serial three-compartment model (outside<==>compartment 1<==>compartment 2). From the initial slopes (some mMsec-1) net uptake and release of about 3 mumolm-2sec-1 Na+ are determined. Surprisingly, but consistent with previous tracer flux measurements (Mummert, H., Gradmann, D. 1991. J. Membrane Biol, 124:255-263), these Na+ fluxes are not accompanied by corresponding changes of the transplasmalemma voltage. [Na+]c is neither affected by the membrane voltage, nor by electrochemical gradients of H+ or Cl- across the plasmalemma, nor by cytoplasmic ATP. The results suggest a powerful vesicular transport system for ions which bypasses the conductance of the plasmalemma. In addition, transient increases of [Na+]c have been observed to take place facultatively during action potentials. The exponential distribution of the amplitudes of these transients (many small and few large peaks) points to local events in the more ore less close vicinity of the Na+ recording electrode. These events are suggested to consist of disruption of endoplasmic vesicles due to a loss of pressure in the cytoplasm.

Electrocoupling of ion transporters in plants.

In the plasmalemma of plants, the major ion transporters are voltage gated. Hence, they are intrinsically coupled via the membrane voltage. Theoretical predictions and electrophysiological recordings on guard cells demonstrate nonlinear oscillations of a dynamic system which provides long-term osmotic adjustment by switching between periods of net uptake and net release of salt, rather than by a steady-state.

Microscopic elements of electrical excitation in Chara: transient activity of Cl- channels in the plasma membrane.

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl- as Ca2+ or Ba2+ (50 or 100 mM), but not as Mg2+ or K+ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl- channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl- channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl- channel activity was favored by voltages positive of the free running membrane voltage (> -45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activation of these channels. Neither type of Cl- channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca2+ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl- channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca2+ and Cl- for triggering and time course: Ca2+ uptake does not activate Cl- channels directly but first supplies a membrane-associated population of Ca2+ storage sites. Depolarization enhances discharge of Ca2+ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca2+ concentration ([Ca2+]cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca2+ buffer system. In turn, the transient rise in [Ca2+]cyt causes the transient activity of those Cl- channels, which are more likely to open at an elevated Ca2+ concentration.