Physiological Voltage Range Research Articles

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

The patch clamp technique has been used to investigate ion permeation and Ca(2+)-dependent gating of a voltage-sensitive Ca2+ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca2+ approximately Sr2+ approximately Ba2+ > Mg2+ >> K+ which is inversely related to the size of the unitary conductances K+ >> Mg2+ approximately Ba2+ > Sr2+ approximately Ca2+, suggesting that ion movement is not independent. In the presence of Ca2+, the unitary K+ current is reduced in a concentration- and voltage-dependent manner by Ca2+ binding at a high affinity site (K0.5 = 0.29 mM at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca2+ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca2+ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca2+ at -41 mV predicts that two Ca2+ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.

The voltage-gated Ca 2+ release channel in the vacuolar membrane of sugar beet resides in two activity states

The voltage-gated Ca 2+ release channel in the vacuolar membrane of sugar beet tap roots resides in two states which show similar reversal potentials in biionic conditions but differ dramatically in their single channel current and open frequency. State I has a unitary Ca 2+ conductance of 3.9 ± 0.5 pS and channel openings are rare at vacuolar resting potentials. After spontaneous and reversible transition into State II the unitary Ca 2+ conductance increases three-fold, accompanied by a concomitant rise in channel activity over the physiological voltage range. The 15- to 19-fold increase in total Ca 2+ current after the State I to State II transition could play an essential role in channel activation during signal transduction.

A cGMP-gated current can control exocytosis at cone synapses

The voltage-gated Ca 2+ current in cone photoreceptors operates over only a small part of the physiological voltage range produced by light and, consequently, appears insufficient for controlling transmitter release. We have used a whole-cell voltage clamp to measure membrane current and the capacitance change produced by exocytosis in solitary cone and rod photoreceptors isolated from the salamander retina. I n both types of photoreceptor, Ca 2+ influx through voltage-gated Ca 2+ channels initiated exocytosis. In addition, Ca 2+ influx through a cGMP-gated channel in the inner segment and synaptic processes of cones also initiated exocytosis. The cGMP-gated current sustained exocytosis over the entire physiological voltage range.

The light-sensitive conductance of hyperpolarizing invertebrate photoreceptors: a patch-clamp study.

Tight-seal recording was employed to investigate membrane currents in hyperpolarizing ciliary photoreceptors enzymatically isolated from the eyes of the file clam (Lima scabra) and the bay scallop (Pecten irradians). These two organisms are unusual in that their double retinas also possess a layer of depolarizing rhabdomeric cells. Ciliary photoreceptors from Lima have a rounded soma, 15-20 microns diam, and display a prominent bundle of fine processes up to 30 microns long. The cell body of scallop cells is similar in size, but the ciliary appendages are modified, forming small spherical structures that protrude from the cell. In both species light stimulation at a voltage near the resting potential gives rise to a graded outward current several hundred pA in amplitude, accompanied by an increase in membrane conductance. The reversal potential of the photocurrent is approximately -80 mV, and shifts in the positive direction by approximately 39 mV when the concentration of extracellular K is increased from 10 to 50 mM, consistent with the notion that light activates K-selective channels. The light-activated conductance increases with depolarization in the physiological range of membrane voltages (-30 to -70 mV). Such outward rectification is greatly reduced after removal of divalent cations from the superfusate. In Pecten, cell-attached recordings were also obtained; in some patches outwardly directed single-channel currents could be activated by light but not by voltage. The unitary conductance of these channels was approximately 26 pS. Solitary ciliary cells also gave evidence of the post stimulus rebound, which is presumably responsible for initiating the "off" discharge of action potentials at the termination of a light stimulus: in patches containing only voltage-dependent channels, light stimulation suppressed depolarization-induced activity, and was followed by a strong burst of openings, directly related to the intensity of the preceding photostimulation.

Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.

An inwardly rectifying K+ current was analysed in isolated toad retinal pigment epithelial (RPE) cells using the perforated-patch clamp technique. The zero-current potential (Vo) of RPE cells averaged -71 mV when the extracellular K+ concentration ([K+]o) was 2 mM. Increasing [K+]o from 0.5 to 5 mM shifted V0 by +43 mV, indicating a relative K+ conductance (TK) of 0.74. At [K+]o greater than 5 mM, TK decreased to 0.53. Currents were larger in response to hyperpolarizing voltage pulses than depolarizing pulses, indicating an inwardly rectifying conductance. Currents were time independent except in response to voltage pulses to potentials positive to 0 mV, where the outward current decayed with an exponential time course. Both the inwardly rectifying current and the transient outward current were eliminated by the addition of 0.5 mM Ba2+, 5 mM Cs+ or 2 mM Rb+ to the extracellular solution. The current blocked by these ions reversed near the K+ equilibrium potential (EK) over a wide range of [K+]o, indicating a highly selective K+ channel. The current-voltage relationship of the isolated K+ current exhibited mild inward rectification at voltages negative to -20 mV and a negative slope conductance at voltages positive to -20 mV. The Cs(+)- and Ba(2+)-induced blocks of the K+ current were concentration dependent but voltage independent. The apparent dissociation constants were 0.8 mM for Cs+ and 40 microM for Ba2+. The K+ conductance decreased when extracellular Na+ was removed. Increasing [K+]o decreased the K+ chord conductance (gK) at negative membrane potentials. In the physiological voltage range, increasing [K+]o from 2 to 5 mM caused gK to decrease by approximately 25%. We conclude that the inwardly rectifying K+ conductance represents the resting K+ conductance of the toad RPE apical membrane. The unusual properties of this conductance may enhance the ability of the RPE to buffer [K+]o changes that take place in the subretinal space at the transition between dark and light.

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells.

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.

Transmitter release and calcium currents at an Aplysia buccal ganglion synapse—II. Modulation by presynaptic receptors

Changes in evoked acetylcholine quantal release induced by histamine, FLRFamide and buccalin were investigated at an identified neuro-neuronal synapse in the buccal ganglion of Aplysia californica. Regulation of acetylcholine release by these neuromodulators was correlated with their actions on the presynaptic Ca 2+ current. We have previously reported that FLRFamide and histamine, respectively, increase and decrease acetylcholine release from buccal neurons B4/B5. Buccalin, a peptide specific to the buccal ganglion, lowered the number of acetylcholine quanta released. Consistent with the synaptic effects, the presynaptic nifedipine-resistant Ca 2+ current that triggers the release of acetylcholine in B4/B5 neurons [Trudeau L.-E. et al. (1993) Neuroscience 53, 571–580] was lowered by buccalin or by histamine and enhanced by FLRFamide. The analysis of tail currents showed that histamine shifts the voltage dependence of the nifedipine-resistant Ca 2+ channels towards more positive voltages, whereas FLRFamide has an opposite action. Buccalin did not affect the voltage dependence of the channels but depressed the amplitude of the Ca 2+ current, an effect which could be due either to a reduction of the number of available Ca 2+ channels, to a decrease of their unitary conductance or to a modification of their gating. Inactivation of presynaptic G proteins prevented the modulatory actions of FLRFamide and histamine on quantal acetylcholine release and also on the voltage dependence of the nifedipine-resistant Ca 2+ channels. This procedure, however, failed to prevent the suppressive effects of buccalin. The possibility of relating the voltage dependence shifts of the Ca 2+ current induced by FLRFamide and histamine to the phosphorylation state of the Ca 2+ channels is discussed. It is concluded that three independent presynaptic pathways initiated by histamine, FLRFamide and buccalin control presynaptic Ca 2+ influx, these modulations being apparent within the physiological range of voltages required to activate Ca 2+ channels.

K+ channels of stomatal guard cells. Characteristics of the inward rectifier and its control by pH.

Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (Ko+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on Ko+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ greater than Rb+ greater than Cs+ much greater than Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (Kd much less than 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of Ko+, and the current activated only with supramillimolar K+ outside (EK+ greater than -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pHo from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pHo, but was blocked reversibly by acid-going intracellular pH. The action of pHo on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less than or equal to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pHo raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pKa approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside.(ABSTRACT TRUNCATED AT 400 WORDS)

A maxi calcium-activated potassium channel from chick lens epithelium

The apical membrane of embryonic chick lens epithelium contains at high density, a large conductance K+ channel whose open probability is increased by Ca++ at the inner surface of the membrane and by depolarization. The conductance of the channel when it is fully open in symmetrical 150 mM K+ solutions is 214 +/- 3 pS (mean +/- std. error). The current through the channel is a function of the K+ concentration. Gating (open probability) at positive transmembrane voltages increases as the internal [Ca++] is raised above 10(-7) M. The open probability decreases monotonically as the transmembrane voltage is made more negative. The channel is at least 87 times more permeable to K+ than to Na+ or Li+ and shows appreciable permeability to Rb+ and NH4+. It has at least three subconductance levels amounting to approximately 3/4, 1/2, and 1/4 the fully open unitary conductance. The occurrence of these subconductance levels is highly variable from one patch to another. The channel is blocked by physiological levels of internal Na+ but not over a physiological voltage range. This block is partially overcome by elevated external K+. This K+ channel from chick lens epithelium is blocked by a number of compounds known to block BK channels in other tissues. Here we show that decamethonium and Ba++ are effective blockers when added to the inner bathing solution at concentrations greater than .1 mM. Tetraethylammonium, Cs+, quinine, quinidine and Ba++ are all effective blockers when applied to the outer side of the channel in the .1 mM - 5 mM range. With the exception of internal Ba++, all of these compounds produce a fast flicker-type blockade. We use a one-site model to quantify the blockade caused by these flicker producing agents. The voltage dependence of the blockade by Cs+ suggests that this channel probably allows multiple occupancy.

Properties of single potassium-selective ionic channels from the apical membrane of rabbit corneal endothelium

The corneal endothelium from several species contains a highly conductive, flickery potassium selective channel in the apical membrane. The channel flicker is not due to blockade. The single channel has an inwardly rectifying current-voltage relationship under symmetrical ionic conditions. Based on reversal potential measurements, it is more than 40:1 selective for potassium over sodium. It also shows subconductance levels. In bathing solutions lacking chloride and bicarbonate, its open probability is less than 0.1 and is quite independent of voltage in the physiological voltage range. Bicarbonate in the bath or DIDs in the pipette increase channel activity. The channel is blocked by external cesium in the 0.5 to 5 mM range. The steepness of the voltage dependence of this blockade is consistent with multiple occupancy.

More Than One Type of Conductance is Activated During Responses of Blowfly Monopolar Neurones

ABSTRACT The principal second-order neurones in the blowfly compound eye, the large monopolar neurones (LMCs), were studied using intracellular recording and discontinuous current-clamp techniques, in combination with measurement of dynamic input resistance. The LMCs had resting potentials of –35 to –45 mV and showed a linear current-voltage relationship in the lamina in the physiological voltage range. The hyperpolarizing light-on transient was associated with a drop in input resistance from 17 ± 5 to 3 ± 1MΩ, and had a reversal potential between –60 and –90 mV. The dynamic input resistance of saturated responses and the properties of reversed responses suggested that more than one conductance was activated during the response of the LMCs. In lamina recordings, the input resistance increased beyond the resting level during repolarization, which can be interpreted in terms of a continuous release of transmitter by the photoreceptor terminals, even in darkness. The input resistance of LMCs in axon recordings in darkness and during the light-on response was generally higher than in the lamina recordings. The responses to light in axons also differed from those recorded in lamina by showing regenerative properties.

Passive membrane properties of motorneurons and their role in long- distance signaling in the nematode Ascaris

In the motornervous system of the large parasitic nematode, Ascaris suum, the dorsal and ventral nerve cords are connected by a repeating pattern of single identified motorneuron processes, called commissures (Stretton et al., 1978). By making microelectrode penetrations of the commissures, we here report the first successful intracellular recordings of nematode neurons. These cells, like muscle cells of Ascaris, exhibit resting potentials of approximately -30 to -40 mV. Several tests indicate that these are the normal resting potentials of the cells and are not low due to damage. Using 2 intracellular microelectrodes (one for stimulation and one for recording), we have determined the input resistance and cable properties of commissural motorneurons. Over the physiological voltage range, the steady-state I-V plots are linear with little indication that voltage-sensitive conductances are contributing substantially to signaling. The membrane capacitance is comparable to that of single biological membranes (range, 0.4-0.9 microF/cm2) and the internal resistivity (range, 79-314 omega cm) is similar to that found in other cells. Because of unusually large membrane resistances (range, 61-251 k omega cm2), the space constants, lambda, are high (range, 4-10 mm). Such membrane properties produce cells that are well-designed for conducting passive signals over long distances. This long-distance signaling ability appears to be due to the intrinsic properties of the motorneuron membrane itself.

Single‐channel analysis of fast transient potassium currents from rat nodose neurones.

Single channels that underlie the fast transient potassium current (IA) were recorded, using patch-clamp techniques, from cultured sensory neurones. The open channel conductance was approximately 22 pS, and was constant over most of the physiological voltage range; single-channel conductance decreased at more depolarized levels. Summing single-channel currents resulted in an average current whose kinetics were similar to the macroscopic IA. The inactivation of these currents, at the potentials we studied, was fitted with a single exponential with a time constant of approximately 30 ms. For the currents evoked by large depolarizing steps (to +40 mV), the mean channel open time equals approximately 30 ms. For currents evoked at less depolarized levels (to 0 mV), the mean open time equals approximately 15 ms, half the inactivation time constant.

Green light (550 nm) inhibits electrogenic Cl− pump in theAcetabularia membrane by permeability increase for the carrier ion

InAcetabularia, the fast, green-light sensitive depolarization (Schilde, C. 1968,Z. Naturforschung23b:1369) has been reexamined. In the physiological voltage range (−170 to −50mV), it appears to be a light-induced current source. The dose-effect relationship is linear up to about 1 A m−2; about 106 photons (550 nm) cause the transfer of one elementary charge across the membrane. Voltage clamp experiments yield the current-voltage relationship of the fast, light-induced pathway over a voltage range from −240 to +200 mV. This current-voltage-relationship does not intersect with the voltage axis up to membrane potentials of +200 mV; it exhibits maximum current at −190 mV (corresponding to the electromotive force of the electrogenic pump, at which its slope conductance is maximum) and shows smaller current under metabolic inhibition by 2,4-dinitrophenol. External cations, including H+, have no significant effect on the fast, light-induced depolarization. The lag between the stimulus and the onset of the reaction is smaller than 40μsec. On the base of actual carrier models for an electrogenic pump, it can be shown by computer simulation that this light-induced pathway reveals the same features as would be expected if the membrane would become permeable for the unoccupied carrier cation in its Cl− binding state.