Apparent Single-channel Conductance Research Articles

Insights into Selectivity Filter Gating of K2P Channels from Single-Channel Recordings

Polymodal K2P channels are regulated by stimuli such as voltage, membrane tension, pH and diverse signalling events. TREK subfamily K2P channels, like some other K+ channels e.g. BK channels, have been shown to gate primarily at the selectivity filter (SF). The TREK-2 single-channel conductance of 200-300 pS makes it accessible to study SF gating at high resolution with exceptional signal to noise ratio. The TREK subfamily in particular is a target for drug discovery because of its involvement in pain perception. Activators of these channels are therefore promising analgesics, through their potential to dampen cellular excitability. Here, we show that applying the K2P channel activator BL1249, in addition to affecting open-probability, increases the apparent single-channel conductance of TREK-2 and we have investigated the various mechanisms which may account for these changes in both conductance and gating. Such studies not only expand our knowledge of the principal mechanisms underlying SF gating of K+ channels but may also help aid our understanding of how some K+ channel agonists exert their effects on the selectivity filter.

Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.

Tight coupling of rubidium conductance and inactivation in human KCNQ1 potassium channels.

KCNQ1 K+ channels in humans are important for repolarization of cardiac action potentials and for K+ secretion in the inner ear. The pore-forming channel subunits form heteromeric complexes with small regulatory subunits of the KCNE family, in particular with KCNE1 to form channels that conduct a slow delayed rectifier K+ current, IKs. This association leads to alteration of biophysical properties, including a slowing of activation, a suppression of inactivation and an increase of the apparent single-channel conductance. In addition, inward Rb+ currents conducted by homomeric KCNQ1 channels are about threefold larger than K+ currents, whereas heteromeric KCNQ1-KCNE1 channels have smaller inward Rb+ currents compared to K+ currents. We determined inactivation properties and compared K+ vs. Rb+ inward currents for channels formed by co-assembly of KCNQ1 with KCNE1, KCNE3 and KCNE5, and for homomeric KCNQ1 channels with point mutations in the pore helix S5 or S6 transmembrane domains. Several of the channels with point mutations eliminated the apparent inactivation of KCNQ1, as described previously (Seebohm et al. 2001). We found that the extent of inactivation and the ratio of Rb+/K+ currents were positively correlated. Since the effect of Rb+ on the current size has been shown previously to be related to a fast 'flickery' process, our results suggest that inactivation of KCNQ1 channels is related to a fast flicker of the open channel. A kinetic model incorporating two open states, no explicit inactivated state and a fast flicker that is different for the two open states is able to account for the apparent inactivation and the correlation of inactivation and large Rb+ currents. We conclude that an association between KCNQ1 and KCNE subunits or removal of inactivation by mutation of KCNQ1 stabilizes the open conformation of the pore principally by altering an interaction between the pore helix and the selectivity filter and with S5/S6 domains.

Estimating Transmitter Release Rates from Postsynaptic Current Fluctuations

A method is presented that allows one to estimate transmitter release rates from fluctuations of postsynaptic current records under conditions of stationary or slowly varying release. For experimental applications, we used the calyx of Held, a glutamatergic synapse, in which "residual current," i.e., current attributable to residual glutamate in the synaptic cleft, is present. For a characterization of synaptic transmission, several postsynaptic parameters, such as the mean amplitude of the miniature postsynaptic current and an apparent single channel conductance, have to be known. These were obtained by evaluating variance and two more higher moments of the current fluctuations. In agreement with Fesce et al. (1986), we found both by simulations and by analyzing experimental records that high-pass filtering of postsynaptic currents renders the estimates remarkably tolerant against nonstationarities. We also found that release rates and postsynaptic parameters can be reliably obtained when release rates are low ( approximately 10 events/msec). Furthermore, during a long-lasting stimulus, the transmitter release at the calyx of Held was found to decay to a low, stationary rate of 10 events/msec after depletion of the "releasable pool" of synaptic vesicles. This stationary release rate is compatible with the expected rate of recruitment of new vesicles to the release-ready pool of vesicles. MiniatureEPSC (mEPSC) size is estimated to be similar to the value of spontaneously occurring mEPSC under this condition.

Gating and Flickery Block Differentially Affected by Rubidium in Homomeric KCNQ1 and Heteromeric KCNQ1/KCNE1 Potassium Channels

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast “flickery/block” process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.

Novel modulation of a nicotinic receptor channel mutant reveals that the open state is stabilized by ethanol.

Ethanol enhances the gating of a family of related ligand-gated ion channels including nicotinic acetylcholine, serotonin type 3, gamma-aminobutyric acid-A, and glycine receptors. This common action may reflect shared molecular and kinetic mechanisms. In all of these receptors, ethanol enhances multichannel currents elicited with low agonist concentrations, but not with high agonist concentrations. A single mutation in the nicotinic receptor beta subunit, betaT263I, causes ethanol to enhance multichannel currents elicited with both low and high acetylcholine concentrations. Based on the ratios of acetylcholine EC50s in the presence and absence of ethanol, this mutant's sensitivity to enhancement is similar to wild type. Ethanol enhancement of betaT263I receptor activation shows no voltage dependence. In the presence of ethanol, the apparent single-channel conductance of the betaT263I receptor is reduced and the apparent channel lifetime is lengthened. Both the 28% increase in maximal current and the 2-fold reduction in EC50 observed at 300 mM ethanol are quantitatively predicted by simulation of a simple kinetic scheme in which ethanol increases by 4-fold the ratio of microscopic opening rate (beta) to closing rate (alpha) for acetylcholine-bound betaT263I receptors. We conclude that ethanol enhancement of betaT263I currents reflects stabilization of its open-channel state relative to agonist-bound closed states. Ethanol effects in wild-type receptors can also be explained by this mechanism.

High-affinity kainate-type ion channels in rat cerebellar granule cells.

1. Patch-clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre-exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration-response data for kainate showed two components. The EC50 value for the high-affinity component (4 microM) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. 2. In ConA-treated granule cells, currents evoked by 10 microM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 microM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 microM La3+. 3. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, BluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. 4. Whole-cell current-voltage curves for kainate-type currents in granule cells were measured and the ratio of the slope conductances at +40 MV and -40 mV was used as an index of rectification. The mean +40 mV/-40 mV ratio determined from thirty-six granule cells was 1.3 +/- 0.1. Spectral density analysis of kainate-evoked whole-cell current noise gave values for the apparent single-channel conductance, gamma(noise), that were on average about 1 pS. 5. To compare further the properties of recombinant kainate channels with the native kainate-type channels in granule cells, we determined EC50 and gamma(noise) values for SYM 2081 in stable cell lines expressing either (GluR6(R) or GluR6(R) and KA2. Co-expression of KA2 with GluR6(R) shifts the EC50 and gamma(noise) values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. 6. The results demonstrate that cerebellar granule cells in culture express functional kainate-type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate-type channels with different, and perhaps variable, subunit composition.

Nicotinic acetylcholine sensitivity of rat petrosal sensory neurons in dissociated cell culture

Using whole-cell, patch-clamp techniques we investigated acetylcholine (ACh) sensitivity of dissociated sensory neurons from rat petrosal ganglia after 4 h–14 days in vitro. In approx. 68% of petrosal neurons (PN; n=109) ACh, applied by fast perfusion or pressure ejection from a `puffer' pipette, caused a rapid depolarization associated with a conductance increase. Under voltage clamp near the resting potential (approx. −60 mV), ACh induced a hexamethonium-sensitive, inward current ( I ACh), mimicked by nicotine application, suggesting the presence of neuronal nicotinic acetylcholine receptors (nAChR). The reversal potential of I ACh occurred near 0 mV ( n=4), a region where the I-V curve displayed a prominent rectification. The dose-response relation for I ACh versus ACh concentration was fitted by the Hill equation with EC 50=approx. 33.9 μM and Hill coefficient=approx. 1.6. The activation phase of I ACh was well fitted by a single exponential with mean (±S.E.M.) time constant of 102±82 ms ( n=6); the desensitization phase of I ACh was best fitted by the sum of two exponentials, with time constant of 870±210 ms ( n=6) and 8576±1435 ms (at −70 mV). Fluctuation analysis yielded an apparent single-channel conductance of 21.6±10 pS (mean±S.E.M.; n=4). These data indicate that a major subpopulation of sensory neurons in visceral petrosal ganglia of the rat express nAChR. Thus, if similar receptors are present on corresponding nerve terminals, they could mediate fast afferent excitation in response to ACh released at peripheral targets, e.g., the chemosensory carotid body.

Ca2+ ion permeability properties of (R,S) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in isolated interneurons from the olfactory bulb of the rat.

The aim of the study was to investigate the divalent cation permeability of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors expressed in interneurons of the olfactory bulb. Kainic acid (KA) was used as agonist to activate AMPA-receptor-mediated currents, which were recorded with the use of the patch-clamp technique. In interneurons acutely isolated from the olfactory bulb, the current responses to KA showed linear/outwardly rectifying current-voltage (I-V) relationships with a positive average reversal potential of +7 mV in normal external medium (1 mM Ca2+, 1 mM Mg2+). Raising the external Ca2+ concentration to 10 mM suppressed the amplitude, whereas omission of Ca2+ enhanced the amplitude of the current. Spectral analysis of the increase in current variance produced by KA indicated that the decreased amplitude observed in 10 mM Ca2+ was accompanied by a reduction in the apparent single-channel conductance. Raising the concentration of Mg2+ from 1 to 10 mM had a weak depressant effect on the KA-evoked current amplitude. No shift in the reversal potential was observed when the concentration of Ca2+ or Mg2+ was changed from 1 to 10 mM. Increasing the external medium concentration of Ca2+ to 60 mM not only further depressed the amplitudes of the KA-evoked currents but also gave a pronounced leftward shift in the average reversal potential to -32 +/- 9 (SE) mV (N = 7). For neurons in primary culture, current responses to KA also showed linear/outwardly rectifying I-V relationships with a positive average reversal potential in normal external medium. Substituting N-methylglucamine for Na+ and increasing the Ca2+ concentration to 10 mM gave a leftward shift in the average reversal potential from +9 +/- 3 mV to -47 +/- 4 mV (N = 11) and caused a marked reduction in the amplitude of the KA-evoked currents at negative potentials. The permeability properties of the studied AMPA receptors were well predicted by the Eyring rate model (symmetrical, 2 barriers, 1 site). The model gave a pCa2+/pK+ permeability ratio of 0.06 for acutely isolated interneurons and 0.14 for interneurons in primary culture. The constant field theory, which failed to successfully reproduce all the experimental data, gave corresponding low permeability ratios of 0.18 and 0.40 for acutely isolated cells and cells in primary culture, respectively. Thus it is concluded that interneurons in the olfactory bulb mainly express AMPA receptors with low permeability to Ca2+ ions.

Hofmeister effect in ion transport: reversible binding of halide anions to the roflamycoin channel

We have studied the anion-dependent gating of roflamycoin ion channels using spectral analysis of noise in currents through multichannel planar lipid bilayers. We have found that in addition to low frequency current fluctuations that may be attributed to channel switching between open and closed conformations, roflamycoin channels exhibit a pronounced higher frequency noise indicating that the open channel conductance has substates with short lifetimes. This noise is well described by a Lorentzian spectrum component with a characteristic cutoff frequency that depends on the type of halide anions according to their position in the Hofmeister series. It is suggested that transitions between the substates correspond to a reversible ionization of the channel by a penetrating anion that binds to the channel structure, more chaotropic anions being bound for longer times. Within a framework of a two-substate model, the duration of the substate with reduced electrostatic barrier for cation current varies exponentially with anion electron polarizability. This explains two features of the roflamycoin channel reported earlier: the increase in apparent single-channel conductance along the series F- < Cl- < Br- < I- and the reverse of channel selectivity from anionic for KF to cationic for KI.

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve.

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.

A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones.

1. The properties of gamma-aminobutyric acidA (GABAA) receptor-ion channel complexes and the interaction with the transition metal zinc, were studied on rat sympathetic and cerebellar neurones in dissociated culture using patch clamp recording techniques. 2. The antagonism of GABA-induced membrane currents by zinc on sympathetic neurones was subject to developmental influence. Using embryonic sympathetic neurones acutely cultured for 24-72 h, GABA responses were more depressed by zinc when compared to responses evoked on adult neurones cultured for the same period. For neurones developing in vivo, the percentage inhibition of GABA responses produced by zinc in embryonic neurones was estimated to decline by 50% after 48.2 days following birth. 3. Embryonic sympathetic neurones maintained in culture for prolonged periods (40-50 days in vitro, DIV) became less sensitive to zinc when compared to neurones cultured for shorter periods (10-20 DIV). The decrease in the zinc inhibition for neurones maintained in vitro proceeded at an apparent rate of 0.55% per day. 4. Activation of the GABA receptor by muscimol (0.2-2 microM) was also antagonized by zinc (50-100 microM). 5. Lowering the pH of the perfusing Krebs solution did not affect the inhibition of GABA responses by zinc on sympathetic neurones. 6. Modulation of the GABAA receptor by some benzodiazepines, a barbiturate, a steroid based on pregnanolone, or antagonists bicuculline and picrotoxinin, did not interfere with the antagonism exerted by zinc on sympathetic neurones. A novel binding site for zinc on the GABAA receptor is proposed. 7. Analysis of the GABA-activated current noise on sympathetic neurones revealed two kinetic components to the power spectra requiring a double Lorentzian fit. The time constant describing the fast component (tau 2, 2.1 ms) was unaffected by zinc, whereas the slow component time constant (tau 1, 21.7 ms) was slightly reduced to 17.1 ms. 8. The apparent single-channel conductance for GABA-activated ion channels was determined from the power spectra (gamma s = 22.7 pS) and also from the relationship between the mean GABA-induced inward current and the variance of the current (gamma v = 24 pS). Zinc (25-100 microM) did not affect the single-channel conductance. 9. Single GABA-activated ion channels were recorded from outside-out patches taken from the soma of large cerebellar neurones. Single GABA channels were capable of activation to multiple current amplitudes which were assessed into the following conductance levels: 8, 18, 23, 29 and 34 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Modes of hexamethonium action on acetylcholine receptor channels in frog skeletal muscle

1. The antagonism between hexamethonium and cholinoceptor agonists was investigated in frog skeletal muscle fibres with voltage-clamp techniques. Hexamethonium caused a voltage-dependent reduction in the amplitude of endplate currents. For neurally evoked endplate currents, the reduction increased e-fold with a 38 mV membrane hyperpolarization. 2. The effect of hexamethonium on the time course of endplate currents was small, and was most apparent as a slight prolongation of the decay phase at hyperpolarized potentials (more negative than -100 mV). A similar small prolongation of single channel lifetime was detected with fluctuation analysis techniques. Hexamethonium produced a voltage-dependent reduction in apparent single channel conductance as the membrane was hyperpolarized. 3. Log (concentration-response) curves for acetylcholine (ACh)-induced currents, determined either from currents accompanying ramp changes in membrane potential or from steady state currents in voltage-jump experiments, were less steep for responses in the presence of hexamethonium. This reduction in slope became more pronounced at more negative membrane potentials. Observations at +50 mV suggested that the equilibrium constant for competitive antagonism was approximately 200 microM. 4. In voltage-jump experiments with a two-microelectrode voltage clamp, the current evoked by ACh in the presence of hexamethonium differed from that recorded with ACh alone. In the presence of hexamethonium, the expected 'instantaneous' ohmic increase in membrane current in response to a hyperpolarizing step was not detected; instead a decrease in current was observed. This problem was further investigated with a vaseline-gap voltage-clamp technique which provides improved temporal resolution. With this method a rapid decrease in the ACh-induced inward current was observed with step hyperpolarizations in the presence of hexamethonium. 5. When the membrane potential was stepped back to its resting level from a more hyperpolarized potential in the presence of hexamethonium, there was a surge of ACh-induced inward current that decayed with a time constant of less than 100 microseconds. 6. The slow relaxation in the ACh-induced current that followed a voltage step recorded in the presence of hexamethonium was slower than that recorded with ACh alone. In the presence of hexamethonium the time constant of this relaxation increased e-fold for a 67 mV hyperpolarization. 7. The results are consistent with a rapid voltage-dependent block of ACh-activated channels by hexamethonium with hyperpolarization, and voltage-dependent unblock with depolarization. The voltagedependent block is combined with competitive antagonism at the ACh receptors. However, not all observations appear to be compatible with a simple sequential block of open ion channels, but rather suggest that occupation of the channel by hexamethonium may not prevent channel closure.

Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells.

Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth.

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (Po) increased with depolarization; Po was 0.5 at -40 to -50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1 nM at -45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in the Po vs. Vm relation such that the channels were more likely to be open at potentials less than 40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.

Non-stationary fluctuations of the potassium conductance at the node of ranvier of the frog.

Potassium currents were recorded from voltage-clamped nodes of isolated, myelinated axons of Rana pipiens. Nodes were maintained in a modified Ringer solution containing tetrodotoxin to block sodium current and 47.5 mM-potassium to minimize effects of extracellular potassium accumulation. Voltage protocols included depolarizing pulses lasting a few milliseconds to several seconds. Fluctuations about the ensemble average of the current were characterized in terms of non-stationary variance and autocovariance. The fluctuations had a Gaussian amplitude distribution and were virtually free of contaminations from systematic variations of the membrane current. Corrections for background noise were based on measurements done while potassium current was blocked with tetraethylammonium, and on simulations of extrinsic current fluctuations expected to arise from noise in the actual membrane voltage. The fluctuations were attributed to variations of nodal potassium conductance, since they were absent at the reversal potential of the potassium current and at membrane voltages that do not activate potassium current. Covariances indicated that voltage steps that reversed a macroscopic potassium current also reversed the sign of the fluctuation. Plots of the conductance variance versus the mean potassium conductance were generated from both the activation and deactivation (tail) phases of the potassium currents at various voltages between -80 and +70 mV. When the current was activated by a small depolarization (-50 mV) the trajectories from both phases were indistinguishable and were fitted by the parabola expected for a single population of channels with only one open-channel conductance. Apparent single-channel conductance from the early activation phase averaged 24 pS and was not significantly voltage dependent. In contrast, experiments with large depolarizations (+10 to +70 mV) gave significantly different variance--mean trajectories during activation and deactivation and these trajectories were poorly fitted by parabola. This result indicates that the fluctuations reflect several populations of channels and/or a population of channels that can have several levels of non-zero conductance. Projections of the fluctuation covariance showed long correlations, as well as the rapidly decaying component expected from the activation gating of channels. A slow fluctuation arose at a time slightly later than the rise of potassium current, spanned the entire length of brief depolarizations, and extended up to 880 ms during long depolarizations.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage-dependent calcium channels from brain incorporated into planar lipid bilayers.

Many important physiological processes, including neurotransmitter release and muscle contraction, are regulated by the concentration of Ca2+ ions in the cell. Levels of cytoplasmic Ca2+ can be elevated by the entry of Ca2+ ions through voltage-dependent channels which are selective for Ca2+, Ba2+ and Sr2+ ions. We have measured currents through single, voltage-dependent calcium channels from rat brain that have been incorporated into planar lipid bilayers. Channel gating was voltage-dependent: membrane depolarization increased the channel open times and decreased the closed times. The channels were selective for divalent cations over monovalent ions. The well-known calcium channel blockers, lanthanum and cadmium, produced a concentration-dependent reduction of the apparent single-channel conductance. Contrary to expectations, the nature of the divalent cation carrying current through the channel affected not only the single-channel conductance, but also the channel open times, with mean open times being shortest for barium.

Physostigmine prolongs the elementary event underlying decay of inhibitory postsynaptic currents in Aplysia

Inhibitory postsynaptic currents (IPSCs) recorded at cholinergic synapses between identified neurons of the buccal ganglia of Aplysia decay exponentially. IPSC decay has been ascribed to a receptor-channel-dependent kinetic process, rather than to changes in subsynaptic acetylcholine (ACh) concentration, but is prolonged by the anticholinesterase eserine. Our data are consistent with a direct effect of 1.6 X 10(-4) M eserine on an elementary channel process. IPSC decay remains single exponential during eserine prolongation, suggesting that eserine slows the kinetic process responsible for decay, rather than substituting an additional kinetic process. In either control sea water or eserine, noise spectra computed from currents induced by pressure ejection of ACh are fitted by the sum of two Lorentzians. The slow corner frequency f1 is reduced from 6.3 to 3.5 Hz by eserine, consistent with a direct eserine action on receptor-channel kinetics, whereas apparent single-channel conductance was unchanged. Both IPSCs and ACh noise recorded in the same cells were comparably slowed by eserine. In eserine, decay time constant tau agreed with noise f1; however, tau and f1 in control sea water differed by 31%. The discrepancy may be accounted for by invoking an additional component to the recorded noise spectra, perhaps produced by synaptically active choline. In addition to the direct effect on kinetics, prolonged exposure to eserine produces a slow extra component to the IPSC decay tail.

Single channel currents from excised patches of muscle membrane.

The currents through single acetylcholine-activated channels were measured on membrane fragments that had been torn from rat muscle myotubes with patch pipettes. The membrane fragments were sealed into the pipette by using the "gigohm-seal" technique of Neher, which also permitted voltage clamp of the membrane via the patch electrode. Membrane patches were excited by sudden withdrawal of the electrode from the cell. Substitution of fluoride for chloride ions in the bathing solution could prevent or reverse the tendency for the membrane at the electrode tip to seal over into a closed vesicle. The single membrane layer at the electrode tip could remain functional for up to 30 min. The apparent single channel conductance was minimally affected by excision. The current-voltage relationships for the single channel currents show that the inside (i.e., cytoplasmic surface) of the membrane fragment was exposed to the bathing solution. In symmetric Na solutions the current-voltage curve was nearly linear and reversed at approximately 0 mV. In other bathing solutions from 40 to 500 mM NaF, the observed zero current potential was close to that predicted by the Nernst equation. We present evidence that internal Na interacts with the channel, causing both saturation of outward current and block of inward current. At + 100 mV the apparent dissociation constant for internal Na was 138 mM.

Voltage-dependent caesium blockade of a cation channel from fragmented sarcoplasmic reticulum

Cs+ ions block the K+ conductance of a voltage-gated ionic channel derived from fragmented sarcoplasmic reticulum (SR) and incorporated into an artificial phospholipid bilayer. The block follows a single-site titration curve with a voltage-dependent dissociation constant of 18 mM at +50 mV. Blockade observed with the macroscopic conductance can be fully accounted for by the reduction of the apparent single-channel conductance in the presence of Cs+. The voltage dependence is explained by assuming that Cs+ binds to the open state of a single channel on a site located about 40% of the way through the membrane from the cis side (the side to which the SR vesicles are added). The blocking site is accessible only from the cis side.