Open channel noise. I. Noise in acetylcholine receptor currents suggests conformational fluctuations.

The random passage of ions through an open channel is expected to result in shot noise fluctuations in the channel current. The patch-clamp technique now allows fluctuations of this size to be observed in single-channel currents. In the experiments reported here the acetylcholine-induced currents in cultured rat muscle cells were analyzed; fluctuations were found that were considerably larger than expected for shot noise. A low-frequency component, which was fitted with a Lorentzian, was examined in detail; it appears to arise from fluctuations in channel conductance of approximately 3% on a time scale of 1 ms. The characteristic relaxation time is voltage dependent and temperature dependent (Q10 approximately equal to 3) suggesting that the fluctuations arise from conformational fluctuations in the channel protein.     

Origins of open-channel noise in the large potassium channel of sarcoplasmic reticulum

Open-channel noise was studied in the large potassium channel of the sarcoplasmic reticulum (SR). Inside-out patches were excised directly from the SR of split skeletal muscle fibers of lobster, with lobster relaxing ringer (LRR) in bath and pipette. The power spectrum of open- channel noise is very low and approximately flat in the 100 Hz-10 kHz frequency range. At 20 degrees C, with an applied voltage of 50 mV, the mean single-channel current (i) is 9 pA (mean single-channel conductance = 180 pS) and the mean power spectral density 1.1 x 10(-29) A2/Hz. The latter increases nonlinearly with (i), showing a progressively steeper dependence as (i) increases. At 20 mV, the mean power spectral density is almost independent of (i) and approximately 1.4 times that of the Johnson noise calculated for the equivalent ideal resistor with zero net current; at 70 mV it increases approximately in proportion to (i)2. The mean power spectral density has a weak temperature dependence, very similar to that of (i), and both are well described by a Q10 of 1.3 throughout the range 3-40 degrees C. Discrete ion transport events are thought to account for a significant fraction of the measured open-channel noise, probably approximately 30-50% at 50 mV. Brief interruptions of the single-channel current, due either to blockage of the open channel by an extrinsic aqueous species, or to intrinsic conformational changes in the channel molecule itself, were a possible additional source of open-channel noise. Experiments in modified bathing solutions indicate, however, that open-channel noise is not affected by any of the identified aqueous species present in LRR. In particular, magnesium ions, the species thought most likely to cause brief blockages, and calcium and hydrogen ions, have no detectable effect. This channel's openings exhibit many brief closings and substrates, due to intrinsic gating of the channel. Unresolved brief full closings are calculated to make a negligible contribution (< 1%) to the measured power spectral density. The only significant source of noise due to band width-limited missed events is brief, frequent 80% substrates (mean duration 20 microseconds, mean frequency 1,000 s-1) which account for a small part of the measured power spectral density (approximately 14%, at 50 mV, 20 degrees C). We conclude that a large fraction of the measured open-channel noise results from intrinsic conductance fluctuations, with a corner frequency higher than the resolution of our recordings, in the range 10(4)-10(7) Hz.(ABSTRACT TRUNCATED AT 400 WORDS)     

Open channel noise. II. A test for coupling between current fluctuations and conformational transitions in the acetylcholine receptor.

The first paper of this series demonstrated that the open-channel currents in the acetylcholine receptors in cultured rat muscle show fluctuations on a time scale of approximately 1 ms. In this paper the hypothesis is tested that these fluctuations are coupled to the gating mechanism that opens and closes the channel. Such a coupling could arise if the channel current and the energy barrier for gating transitions both showed fluctuations having a common origin such as a motion of part of the receptor molecule. A test for coupled fluctuations is made by averaging approximately 1,000 channel opening or closing transitions to search for the small relaxation in the current that is predicted. At a resolution of approximately 1% of the single-channel current amplitude, no such relaxation is observed. It is concluded that any coupled fluctuations are small; fluctuations in the energy barrier for the open-closed conformational transition must be smaller than about 0.3 kT.     

Multimodal action of single Na+ channels in myocardial mouse cells.

Unitary Na+ currents of myocardial mouse cells were studied at room temperature in 10 cell-attached patches, each containing one and only one channel. Small-pore patch pipettes (resistance 10-97 M omega when filled with 200% Tyrode's solution) with exceptionally thick walls were used. Observed were both rapidly inactivating (6 patches) and slowly inactivating (3 patches) Na+ currents. In one patch, a slow transition from rather fast to slow inactivation was detected over a time of 0.5 h. A short and a long component of the open-channel life time were recorded at the beginning, but only a short one at the end of the experiment. Concomitantly, the first latency was slowed. Amplitude histograms showed that the electrochemical driving force across the pore of the channel did not change during this time. In three patches, a fast and repetitive switching between different modes of Na+ channel action could be clearly identified by plotting the long-time course of the averaged current per trace. The ensemble-averaged current formed in each mode was different in kinetics and amplitude. Each mode had a characteristic mean open-channel life time and distribution of first latency, but the predominant single-channel current amplitude was unaffected by mode switches. It is concluded that two types of changes in kinetics may happen in a single Na+ channel: fast and reversible switches between different modes, and a slow loss of inactivation.     

Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state

Swelling-induced loss of organic osmolytes from cells is mediated by an outwardly rectified, volume-sensitive anion channel termed VSOAC (Volume-Sensitive Organic osmolyte/Anion Channel). Similar swelling- activated anion channels have been described in numerous cell types. The unitary conductance and gating kinetics of VSOAC have been uncertain, however. Stationary noise analysis and single-channel measurements have produced estimates for the unitary conductance of swelling-activated, outwardly rectified anion channels that vary by > 15-fold. We used a combination of stationary and nonstationary noise analyses and single-channel measurements to estimate the unitary properties of VSOAC. Current noise was analyzed initially by assuming that graded changes in macroscopic current were due to graded changes in channel open probability. Stationary noise analysis predicts that the unitary conductance of VSOAC is approximately 1 pS at 0 mV. In sharp contrast, nonstationary noise analysis demonstrates that VSOAC is a 40-50 pS channel at +120 mV (approximately 15 pS at 0 mV). Measurement of single-channel events in whole-cell currents and outside- out membrane patches confirmed the nonstationary noise analysis results. The discrepancy between stationary and nonstationary noise analyses and single-channel measurements indicates that swelling- induced current activation is not mediated by a graded increase in channel open probability as assumed initially. Instead, activation of VSOAC appears to involve an abrupt switching of single channels from an OFF state, where channel open probability is zero, to an ON state, where open probability is near unity.     

Single-Channel Properties of IKs Potassium Channels

Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K⁺, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz–20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very “flickery” single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.     

Mechanotransduction by Membrane Proteins: The speed of the hair cell mechanotransducer channel revealed by fluctuation analysis

Although mechanoelectrical transducer (MET) channels have been extensively studied, uncertainty persists about their molecular architecture and single-channel conductance. We made electrical measurements from mouse cochlear outer hair cells (OHCs) to reexamine the MET channel conductance comparing two different methods. Analysis of fluctuations in the macroscopic currents showed that the channel conductance in apical OHCs determined from nonstationary noise analysis was about half that of single-channel events recorded after tip link destruction. We hypothesized that this difference reflects a bandwidth limitation in the noise analysis, which we tested by simulations of stochastic fluctuations in modeled channels. Modeling indicated that the unitary conductance depended on the relative values of the channel activation time constant and the applied low-pass filter frequency. The modeling enabled the activation time constant of the channel to be estimated for the first time, yielding a value of only a few microseconds. We found that the channel conductance, assayed with both noise and recording of single-channel events, was reduced by a third in a new deafness mutant, Tmc1 p.D528N. Our results indicate that noise analysis is likely to underestimate MET channel amplitude, which is better characterized from recordings of single-channel events.     

Open channel noise. VI. Analysis of amplitude histograms to determine rapid kinetic parameters.

Recently we reported that rapid fluctuations of ion currents flowing through open gramicidin A channels exceed the expected level of pure transport noise at low ion concentrations (Heinemann, S. H. and F. J. Sigworth. 1990. Biophys. J. 57:499-514). Based on comparisons with kinetic ion transport models we concluded that this excess noise is likely caused by current interruptions lasting approximately 1 microsecond. Here we introduce a method using the higher-order cumulants of the amplitude distribution to estimate the kinetics of channel closing events far below the actual time resolution of the recording system. Using this method on data recorded with 10 kHz bandwidth, estimates for gap time constants on the order of 1 microsecond were obtained, similar to the earlier predictions.     

Correlation analysis of electrical noise in lipid bilayer membranes: kinetics of gramicidin A channels.

If a membrane contains ion-conducting channels which form and disappear in a random fashion, an electric current which is passed through the membrane under constant voltage shows statistical fluctuations. Information on the kinetics of channel formation and on the conductance of the single channel may be obtained by analyzing the electrical noise generated in a membrane containing a great number of channels. For this purpose the autocorrelation function of the current noise is measured at different concentrations of the channel-forming substance. As a test system for the application of this technique we have used lipid bilayer membranes doped with gramicidin A. From the correlation time of the current noise generated by the membrane, the rate constants of formation (k-R) and dissociation (k-D) of the channels could be determined. In addition, the mean square of the current fluctuations yielded the single-channel conductance lambda. The values of k-R, k-D, and lambda obtained from the noise analysis agreed closely with the values determined by relaxation measurments and single-channel experiments.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block.

K+-selective ion channels from mammalian sarcoplasmic reticulum were inserted into planar phospholipid bilayers, and single-channel currents measured in solutions containing Cs+. Current through this channel can be observed in symmetrical solutions containing only Cs+ salts. At zero voltage, the Cs+ conductance is approximately 15-fold lower than the corresponding K+ conductance. The open channel rectifies strongly in symmetrical Cs+ solutions, and the Cs+ currents are independent of Cs+ concentration in the range 18-600 mM. Biionic (Cs+/K+) reversal potentials are only 10 mV, showing that Cs+ is nearly as permeant as K+, though much less conductive. Addition of Cs+ to symmetrical K+ solutions reduces current through the channel in a voltage-dependent way. The results can be explained by a free energy profile in which the channel's selectivity filter acts in two ways: to provide binding sites for the conducting ions and to serve as a major rate-determining structure. According to this picture, the main difference between high-conductance K+ and low-conductance Cs+ is that Cs+ binds to an asymmetrically positioned site approximately 20-fold more tightly than does K+.     

Cav1.4 encodes a calcium channel with low open probability and unitary conductance

When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of -20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was approximately 4 pS compared with approximately 20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of approximately 21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by approximately 45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.     

Marcroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidianCiona intestinalis

Summary Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidianCiona intestinalis to investigate the properties of two voltage-dependent Ca²⁺ currents found in this cell. The peak of the low threshold current (channel I) occurred at –20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca²⁺ and Mg²⁺ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during changel I openings were recorded when micromolar concentrations of Ca²⁺ or Mg²⁺ were added to the patch pipette solutions. Slopes of the blocking rate constantvs. concentration gave binding constants of 6.4×10⁶ m ^–1 sec^–1 for Mg²⁺ and 4.5×10⁸ m ^–1 sec^–1 for Ca²⁺. The Ca²⁺ block was somewhat relieved at negative potentials, whereas the Mg²⁺ block was not, suggesting that Ca²⁺, but not Mg²⁺, can exit from the binding site toward the cell interior.     

Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.     

Noise analysis of ion channels in non-space-clamped cables: estimates of channel parameters in olfactory cilia.

Ion channels in the cilia of olfactory neurons are part of the transduction machinery of olfaction. Odorant stimuli have been shown to induce a biphasic current response, consisting of a cAMP-activated current and a Ca(2+)-activated Cl- current. We have developed a noise analysis method to study ion channels in leaky cables, such as the olfactory cilium, under non-space-clamp conditions. We performed steady-state noise analysis on ligand-induced currents in excised cilia, voltage-clamped at input and internally perfused with cAMP or Ca2+. The cAMP-activated channels analyzed by this method gave results similar to those of single-channel recordings (gamma = 8.3 pS). Single-channel currents have not yet been recorded for the Ca(2+)-activated Cl- channels. Using our noise analysis method, we estimate a unit conductance, gamma = 0.8 pS, for these channels. The density of channels was found to be approximately 70 channels/micron2 for both channel species.     

Trapping of an open-channel blocker at the frog neuromuscular acetylcholine channel.

At the ganglionic nicotinic acetylcholine channel (Gurney, A. M., and H. P. Rang, 1984, Br. J. Pharmacol., 82:623-642) and on some cholinergic neuromuscular synapses of Crustacea (Lingle, C., 1983a, J. Physiol. (Lond.), 339:395-417; Lingle, C., 1983b, J. Physiol. (Lond.), 339:419-437), some agents that block cholinergic currents by an open-channel block mechanism appear to become trapped within the channel when it subsequently closes. It is unknown whether trapping of some open-channel blockers might also occur at the neuromuscular nicotinic acetylcholine channel. Here we show that the long-lived cholinergic blocking action of chlorisondamine, a ganglionic nicotinic blocker, can in part be most simply explained by an open-channel block mechanism followed by a subsequent trapping of the blocking molecule within the closed ion channel. Unique structural characteristics of the chlorisondamine molecule place several provocative constraints on the mechanism by which trapping may be occurring.     

The conductance of the muscle nicotinic receptor channel changes rapidly upon gating.

We have recorded single-channel currents through fetal-type muscle nicotinic receptor channels at recording bandwidths of approximately 50 and 75 kHz. The time course of the rising phase of aligned and averaged openings can be entirely accounted for if it is assumed that the conductance of the single channel changes instantaneously, and that alignment and averaging introduce a dispersion of 2-3 microseconds. We conclude that we find no evidence for a gradual change in conductance as a channel opens or closes. The shapes of averaged power spectra are consistent with this conclusion, insofar as they exclude an exponential relaxation in the transition with a time constant of 10 microseconds or more.     

Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.     

Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches

We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel records. Teh lifetime of the open channel at - 40 mV was increased about 10-fold by NBA treatment without affecting the amplitude of single-channel currents. A binomial analysis was used both before and after treatment to determine the number of channels within the excised patch. NBA was shown to have little effect on activation kinetics, as determined by an examination of both the rising phase of averaged currents and measurements f the delay between the start of the pulse and the first channel opening. Our data support a kinetic model of sodium channel activation in which the rate constant leading back from the open state to the last closed state is slower than expected from a strict Hodgkin-Huxley model. The data also suggest that the normal open-channel lifetime is primarily determined by the inactivation process in the voltage range we have examined.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.