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.     

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.     

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.     

Ion channels activated by L-glutamate and GABA in cultured cerebellar neurons of the rat

Glutamate and GABA-receptor channels were investigated in explants of rat cerebellum grown in cell culture. The patch-clamp technique was used to examine neurons under whole cell clamp and the properties of channels were derived by analysis of glutamate and GABA-evoked current noise. In addition, single channel currents activated by glutamate were recorded from isolated outside-out patches of membrane. We found evidence for at least two types of glutamate receptor-channels in cerebellar cells. Some neurons exhibited a channel of 50 pS conductance with a Lorentzian noise spectrum of 5.9 ms time constant. Single channels were readily resolved both in whole cell clamp and excised patches. Other neurons possessed low conductance channels which produced two component spectra. Estimates of the single channel conductance gave a value of about 140 fS. GABA channel noise obtained from these cells was also fitted by two component spectra which gave single channel conductance of 16 pS.     

Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni(2+)

We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na(+) channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance (C(m)) measurements and noise analysis to evaluate the effects of cAMP and Ni(2+) on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker-channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current (I(ami)) and conductance (G(ami)). Furthermore, C(m) was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni(2+) (2.5 mM) inhibited about 60% of the rENaC current and conductance while C(m) remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni(2+) on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.     

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.     

Conductivity noise in transmembrane ion channels due to ion concentration fluctuations via diffusion.

A Green's function approach is developed from first principles to evaluate the power spectral density of conductance fluctuations caused by ion concentration fluctuations via diffusion in an electrolyte system. This is applied to simple geometric models of transmembrane ion channels to obtain an estimate of the magnitude of ion concentration fluctuation noise in the channel current. Pure polypeptide alamethicin forms stable ion channels with multiple conductance states in artificial phospholipid bilayers isolated onto tips of micropipettes with gigaohm seals. In the single-channel current recorded by voltage-clamp techniques, excess noise was found after the background instrumental noise and the intrinsic Johnson and shot noises were removed. The noise que to ion concentration fluctuations via diffusion was isolated by the dependence of the excess current noise on buffer ion concentration. The magnitude of the concentration fluctuation noise derived from experimental data lies within limits estimated using our simple geometric channel models. Variation of the noise magnitude for alamethicin channels in various conductance states agrees with theoretical prediction.     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

Elementary Functional Properties of Single HCN2 Channels

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are tetramers that evoke rhythmic electrical activity in specialized neurons and cardiac cells. These channels are activated by hyperpolarizing voltage, and the second messenger cAMP can further enhance the activation. Despite the physiological importance of HCN channels, their elementary functional properties are still unclear. In this study, we expressed homotetrameric HCN2 channels in Xenopus oocytes and performed single-channel experiments in patches containing either one or multiple channels. We show that the single-channel conductance is as low as 1.67 pS and that channel activation is a one-step process. We also observed that the time between the hyperpolarizing stimulus and the first channel opening, the first latency, determines the activation process alone. Notably, at maximum hyperpolarization, saturating cAMP drives the channel to open for unusually long periods. In particular, at maximum activation by hyperpolarization and saturating cAMP, the open probability approaches unity. In contrast to other reports, no evidence of interchannel cooperativity was observed. In conclusion, single HCN2 channels operate only with an exceptionally low conductance, and both activating stimuli, voltage and cAMP, exclusively control the open probability.     

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.     

Elementary Functional Properties of Single HCN2 Channels

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are tetramers that evoke rhythmic electrical activity in specialized neurons and cardiac cells. These channels are activated by hyperpolarizing voltage, and the second messenger cAMP can further enhance the activation. Despite the physiological importance of HCN channels, their elementary functional properties are still unclear. In this study, we expressed homotetrameric HCN2 channels in Xenopus oocytes and performed single-channel experiments in patches containing either one or multiple channels. We show that the single-channel conductance is as low as 1.67 pS and that channel activation is a one-step process. We also observed that the time between the hyperpolarizing stimulus and the first channel opening, the first latency, determines the activation process alone. Notably, at maximum hyperpolarization, saturating cAMP drives the channel to open for unusually long periods. In particular, at maximum activation by hyperpolarization and saturating cAMP, the open probability approaches unity. In contrast to other reports, no evidence of interchannel cooperativity was observed. In conclusion, single HCN2 channels operate only with an exceptionally low conductance, and both activating stimuli, voltage and cAMP, exclusively control the open probability.     

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.     

Low single channel conductance of the major skeletal muscle chloride channel, ClC-1.

We expressed the skeletal muscle chloride channel, ClC-1, in HEK293 cells and investigated it with the patch-clamp technique. Macroscopic properties are similar to those obtained after expression in Xenopus oocytes, except that faster gating kinetics are observed in mammalian cells. Nonstationary noise analysis revealed that both rat and human ClC-1 have a low single channel conductance of about 1 pS. This finding may explain the lack of single-channel data for chloride channels from skeletal muscle despite its high macroscopic chloride conductance.     

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.     

Structural aspects of the sarcoplasmic reticulum K+ channel revealed by gallamine block.

We have studied single-channel conductance fluctuations of K+ channels present in the sarcoplasmic reticulum (SR) membrane systems of rabbit cardiac and skeletal muscle. K+ conductance through the channels is reversibly blocked by gallamine. Conductance block occurs only from the trans side of the channel and is resolved as a smooth reduction in the open state conductance. At a fixed K+ concentration, conduction decreases with increasing gallamine concentration and the data can be fitted to a single-site inhibition scheme. The degree of block seen at a constant gallamine concentration decreases as K+ concentration is increased, indicating competition between gallamine and K+. Gallamine block is voltage dependent, the degree of block increasing with increasing negative holding potential. Quantitative analysis of block yields a zero voltage dissociation constant of 55.3 +/- 16 microM and an effective valence of block of 0.93 +/- 0.12. We conclude that gallamine blocks by interacting with a site or sites located at an electrical distance 30-35% into the voltage drop from the trans side of the channel. This site must have a cross-sectional area of at least 1.2 nm2. The results of this study have been used to modify and extend our view of the structure of the channel's conduction pathway.     

Noise analysis of the glutamate-activated current in photoreceptors.

The glutamate-activated current in photoreceptors has been attributed both to a sodium/glutamate transporter and to a glutamate-activated chloride channel. We have further studied the glutamate-activated current in single, isolated photoreceptors from the tiger salamander using noise analysis on whole-cell patch-clamp recordings. In cones, the current is generated by chloride channels with a single-channel conductance of 0.7 pS and an open lifetime of 2.4 ms. The number of channels per cell is in the range of 10,000-20,000. Activation of the channels requires the presence of both glutamate and sodium. The single-channel conductance and the open lifetime of the channel are independent of the external concentration of glutamate and sodium. External glutamate and sodium affect only the opening rate of the channels. D,L-Threo-3-hydroxyaspartate (THA), a glutamate-transport blocker, is shown to be a partial agonist for the channel. The single-channel conductance is the same regardless of whether glutamate or THA is the ligand, but the open lifetime of the channel is only 0.8 ms with THA as ligand. The glutamate-activated current in rods has a similar single-channel conductance (0.74 pS) and open lifetime (3 ms). We propose a kinetic model, consistent with these results, to explain how a transporter can simultaneously act both as a sodium/glutamate-gated chloride channel and a glutamate/sodium cotransporter.     

Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.     

Recovery of mechano-electrical transduction in rat cochlear hair bundles after postnatal destruction of the stereociliar cross-links

Mechano-electrical transduction (MET) in the stereocilia of outer hair cells (OHCs) was studied in newborn Wistar rats using scanning electron microscopy to investigate the stereociliar cross-links, Nomarski laser differential interferometry to investigate stereociliar stiffness and by testing the functionality of the MET channels by recording the entry of fluorescent dye, FM1-43, into stereocilia. Preparations were taken from rats on their day of birth (P0) or 1–4 days later (P1–P4). Hair bundles developed from the base to the apex and from the inner to outer OHC rows. MET channel responses were detected in apical coil OHCs on P1. To study the possible recovery of MET after disrupting the cross-links, the same investigations were performed after the application of Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and allowing the treated samples to recover in culture medium for 0–20 h. We found that the structure and function were abolished by BAPTA. In P0–P1 samples, structural recovery was complete and the open probability of MET channels reached control values. In P3–P4 samples, complete recovery only occurred in OHCs of the outermost row. Although our results demonstrate an enormous recovery potential of OHCs in the postnatal period, the structural component restricts the potential for therapy in patients.