Quaternary ammonium compounds as structural probes of single batrachotoxin-activated Na+ channels

Quaternary ammonium (QA) blockers are well-known structural probes for studying the permeation pathway of voltage-gated K+ channels. In this study we have examined the effects of a series of n-alkyl-trimethylammonium compounds (Cn-QA) on batrachotoxin (BTX)-activated Na+ channels from skeletal muscle incorporated into planar lipid bilayers. We found that these amphipathic QA compounds (Cn-QA where n = 10-18) block single Na+ channels preferentially from the internal side with equilibrium dissociation constants (KD) in the submicromolar to micromolar range. External application of amphipathic QA compounds is far less effective, by a factor of greater than 200. The block can be described by a QA molecule binding to a single site in the Na+ channel permeation pathway. QA binding affinity is dependent on transmembrane voltage with an effective valence (delta) of approximately 0.5. QA dwell times (given as mean closed times, tau c) increase as a function of n-alkyl chain length, ranging from approximately 13 ms for C10-QA to 500 ms for C18-QA at +50 mV. The results imply that there is a large hydrophobic region within the Na+ channel pore which accepts up to 18 methylene groups of the Cn-QA cation. This hydrophobic domain may be of clinical significance since it also interacts with local anesthetics such as cocaine and mepivacaine. Finally, like BTX-activated Na+ channels in bilayers, unmodified Na+ channels in GH3 cells are also susceptible to QA block. Amphipathic QA cations elicit both tonic and use-dependent inhibitions of normal Na+ currents in a manner similar to that of local anesthetic cocaine. We conclude that amphipathic QA compounds are valuable structural probes to study the permeation pathway of both normal and BTX-activated Na+ channels.     

Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore

We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.     

Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells

Single channel currents through Ca2+-activated K+ channels of bovine chromaffin cells were measured to determine the effects of small ions on permeation through the channel. The channel selects strongly for K+ over Na+ and Cs+, and Rb+ carries a smaller current through the channel than K+. Tetraethylammonium ion (TEA+) blocks channel currents when applied to either side of the membrane; it is effective at lower concentrations when applied externally. Millimolar concentrations of internal Na+ reduce the average current through the channel and produce large fluctuations (flicker) in the open channel currents. This flickery block is analyzed by a new method, amplitude distribution analysis, which can measure block and unblock rates in the microsecond time range even though individual blocking events are not time-resolved by the recording system. The analysis shows that the rate of block by Na+ is very voltage dependent, but the unblock rate is voltage independent. These results can be explained easily by supposing that current flow through the channel is diffusion limited, a hypothesis consistent with the large magnitude of the single channel current.     

External barium block of Shaker potassium channels: evidence for two binding sites

External barium ions inhibit K+ currents of Xenopus oocytes expressing ShH4 delta 6-46, the non-inactivating deletion of the Shaker K+ channel. At the macroscopic level, Ba2+ block comprises both a fast and a slow component. The fast component is less sensitive to Ba2+ (apparent dissociation constant at 0 mV, K(0), approximately 19.1 mM) than the slow component and is also less voltage dependent (apparent electrical distance, delta, approximately 0.14). The slow component (K(0), approximately 9.4 mM, delta approximately 0.25) is relieved by outward K+ current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast component of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unblocking reaction. In contrast, the slow component appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (tau) produced by 2 mM Ba2+ is approximately 159 s (holding potential, HP = -90 mV). However, the blocking rate of this slow component is not a linear function of external Ba2+ and tends to saturate at higher concentrations. This is inconsistent with a simple bi-molecular blocking reaction. These features of external Ba2+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow component of block.     

Characteristics of cocaine block of purified cardiac sarcoplasmic reticulum calcium release channels.

We have examined the effects of cocaine on the SR Ca2+ release channel purified from canine cardiac muscle. Cocaine induced a flicker block of the channel from the cytoplasmic side, which resulted in an apparent reduction in the single-channel current amplitude without a marked reduction in the single-channel open probability. This block was evident only at positive holding potentials. Analysis of the block revealed that cocaine binds to a single site with an effective valence of 0.93 and an apparent dissociation constant at 0 mV (Kd(0)) of 38 mM. The kinetics of cocaine block were analyzed by amplitude distribution analysis and showed that the voltage and concentration dependence lay exclusively in the blocking reaction, whereas the unblocking reaction was independent of both voltage and concentration. Modification of the channel by ryanodine dramatically attenuated the voltage and concentration dependence of the on rates of cocaine block while diminishing the off rates to a lesser extent. In addition, ryanodine modification changed the effective valence of cocaine block to 0.52 and the Kd(0) to 110 mM, suggesting that modification of the channel results in an alteration in the binding site and its affinity for cocaine. These results suggest that cocaine block of the SR Ca2+ release channel is due to the binding at a single site within the channel pore and that modification of the channel by ryanodine leads to profound changes in the kinetics of cocaine block.     

Block of current through single calcium channels by Fe, Co, and Ni. Location of the transition metal binding site in the pore

The blocking actions of Fe2+, Co2+, and Ni2+ on unitary currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCl2 produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarization speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni2+ differs from that produced by Fe2+ and Co2+ because Ni2+ enters the channel approximately 20 times more slowly and exits approximately 50 times more slowly. Ni2+ also differs from the other transition metals because at millimolar concentrations it reduces the amplitude of the unitary current in a concentration-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size.     

Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence approximately 5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QA(C10)) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider ( approximately 6 A) than the ion selectivity filter ( approximately 3 A). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QA(C10) should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.     

Effects of phospholipid surface charge on ion conduction in the K+ channel of sarcoplasmic reticulum.

Single-channel K+ currents through sarcoplasmic reticulum K+ channels were compared after reconstitution into planar bilayers formed from neutral or negatively charged phospholipids. In neutral bilayers, the channel conductance saturates with K+ concentration according to a rectangular hyperbola, with half-saturation at 40 mM K+, and maximum conductance of 220 pS. In negatively charged bilayers (70% phosphatidylserine/30% phosphatidylethanolamine), the conductance is, at a given K+ concentration, higher than in neutral bilayers. This effect of negative surface charge is increasingly pronounced at lower ionic strength. The maximum conductance at high K+ approaches 220 pS in negative bilayers, and the channel's ionic selectivity is unaffected by lipid charge. The divalent channel blocker " bisQ11 " causes discrete blocking events in both neutral and negatively charged bilayers; the apparent rate constant of blocking is sensitive to surface charge, while the unblocking rate is largely unaffected. Bilayers containing a positively charged phosphatidylcholine analogue led to K+ conductances lower than those seen in neutral bilayers. The results are consistent with a simple mechanism in which the local K+ concentration sensed by the channel's entryway is determined by both the bulk K+ concentration and the bulk lipid surface potential, as given by the Gouy-Chapman model of the electrified interface. To be described by this approach, the channel's entryway must be assumed to be located 1-2 nm away from the lipid surface, on both sides of the membrane.     

Measuring the length of the pore of the sheep cardiac sarcoplasmic reticulum calcium-release channel using related trimethylammonium ions as molecular calipers.

After incorporation of purified sheep cardiac Ca(2+)-release channels into planar phospholipid bilayers, we have investigated the blocking effects of a series of monovalent (CH3-(CH2)n-1-N+(CH3)3) and divalent ((CH3)3N(+)-(CH2)n-N+(CH3)3) trimethylammonium derivatives under voltage clamp conditions. All the compounds tested produce voltage-dependent block from the cytoplasmic face of the channel. With divalent (Qn) derivatives the effective valence of block decreases with increasing chain length, reaching a plateau with a chain length of n > or = 7. No decline in effective valence is observed with the monovalent (Un) derivatives. A plausible interpretation of this phenomena suggests that for the 90% of the voltage drop measured, the increase in length following the addition of a CH2 in the chain spans 12.7% of the electrical field. Extrapolating this distance to include the remaining 10% suggests that the applied holding potential falls over a total distance of 10.4 A. In addition, at high positive holding potentials there is evidence for permeation of the trimethylammonium ions and a valency specific relief of block.     

Temperature dependence of drug blockade of a calcium-dependent potassium channel in cultured hippocampal neurons.

The temperature dependence of drug blockade of a calcium-dependent potassium channel K(Ca) has been studied in cultured CA1 hippocampal neurons. Channel openings from a 70-pS K+ channel were recorded when inside-out patches were exposed to a bath solution containing 140 mM K+ and 0.2 mM Ca2+. The mean open times of channel events were not significantly altered when the bath temperature was lowered from 24 degrees to 14 degrees C (Q10 = 1.2). Introduction of the drug RP-62719 into the bath solution (at 5 microM) resulted in the mean open time of the K(Ca) channel to be diminished by 85% (at 24 degrees C) with no change in the amplitudes of the unitary currents. Over the same temperature range of 24 degrees to 14 degrees C, in the presence of RP-62719, the mean open times were significantly prolonged (Q10 = 2.2). A simple open channel block scheme was used to determine the temperature dependence of the onward- (blocking) and off- (unblocking) rate constants. Thermodynamic analysis, using transition rate theory, showed that the blocking rate constant was associated with a large increase in entropy. The relatively high temperature dependence for channel blockade is not consistent with a rate-limiting process established by simple diffusion of the agent to a channel blocking site. Channel block may involve conformational changes in the channel protein as a consequence of hydrophobic interactions between drug and channel sites.     

Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte

The permeation properties of the 147-pS Ca2+-activated K+ channel of the taenia coli myocytes are similar to those of the delayed rectifier channel in other excitable membranes. It has a selectivity sequence of K+ 1.0 greater than Rb+ 0.65 greater than NH4+ 0.50. Na+, Cs+, Li+, and TEA+ (tetraethylammonium) are impermeant. Internal Na+ blocks K+ channel in a strongly voltage-dependent manner with an equivalent valence (zd) of 1.20. Blockade by internal Cs+ and TEA+ is less voltage dependent, with d of 0.61 and 0.13, and half-blockage concentrations of 88 and 31 mM, respectively. External TEA+ is about 100 times more effective in blocking the K+ channel. All these findings suggest that the 147-pS Ca2+-activated K+ channel in the taenia myocytes, which functions physiologically like the delayed rectifier, is the single-channel basis of the repolarizing current in an action potential.     

External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.

The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.     

Mechanism of rectification in inward-rectifier K+ channels

Rectification in inward-rectifier K+ channels is caused by the binding of intracellular cations to their inner pore. The extreme sharpness of this rectification reflects strong voltage dependence (apparent valence is approximately 5) of channel block by long polyamines. To understand the mechanism by which polyamines cause rectification, we examined IRK1 (Kir2.1) block by a series of bis-alkyl-amines (bis-amines) and mono-alkyl-amines (mono-amines) of varying length. The apparent affinity of channel block by both types of alkylamines increases with chain length. Mutation D172N in the second transmembrane segment reduces the channel's affinity significantly for long bis-amines, but only slightly for short ones (or for mono-amines of any length), whereas a double COOH-terminal mutation (E224G and E299S) moderately reduces the affinity for all bis-amines. The apparent valence of channel block increases from approximately 2 for short amines to saturate at approximately 5 for long bis-amines or at approximately 4 for long mono-amines. On the basis of these and other observations, we propose that to block the channel pore one amine group in all alkylamines tested binds near the same internal locus formed by the COOH terminus, while the other amine group of bis-amines, or the alkyl tail of mono-amines, "crawls" toward residue D172 and "pushes" up to 4 or 5 K+ ions outwardly across the narrow K+ selectivity filter. The strong voltage dependence of channel block therefore reflects the movement of charges carried across the transmembrane electrical field primarily by K+ ions, not by the amine molecule itself, as K+ ions and the amine blocker displace each other during block and unblock of the pore. This simple displacement model readily accounts for the classical observation that, at a given concentration of intracellular K+, rectification is apparently related to the difference between the membrane potential and the equilibrium potential for K+ ions rather than to the membrane potential itself.     

Conduction and block by organic cations in a K+-selective channel from sarcoplasmic reticulum incorporated into planar phospholipid bilayers

A collection of organic cations has been used to probe the gross structural features of the ionic diffusion pathway in a K+-selective channel from sarcoplasmic reticulum (SR). Channels were incorporated into planar phospholipid bilayer membranes, and single-channel currents were measured in the presence of ammonium-derived cations in the aqueous phases. Small monovalent organic cations are able to permeate the channel: the channel conductance drops sharply for cations having molecular cross sections larger than 18-20 A2. Impermeant or poorly permeant cations such as tetraethylammonium, choline, and glucosamine, among others, block K+ conduction through the channel. This block is voltage dependent and can be described by a one-site, one-ion blocking scheme. 19 monovalent organic cations blocks primarily from the trans side of the membrane (the side defined as zero voltage), and much more weakly, if at all, from the cis side (to which SR vesicles are added). These blockers all appear to interact with a site located at 63% (average value) of the electric potential drop measured from the trans side. Furthermore, block by 1,3-bis[tris(hydroxymethyl)-methylamino] propane (BTP) shows that the presence of a blocking ion increases the duration of the apparent open state, as expected for a scheme in which the blocking site can be reached only when the channel is open. The results lead to a picture of the channel containing a wide (at least 50 A2) nonselective trans entry in series with a narrow (20 A2) constriction.     

Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that of ruthenium red. These compounds inhibited the open channel with lower affinities (Kd = 170 microM, 4APd; Kd = 656 microM, 4APt), and had Hill coefficients of close to 1. The results suggest that ruthenium red block of the ryanodine receptor is due to binding to multiple sites located in the conduction pore of the channel.     

Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.     

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.     

Methadone block of K+ current in squid giant fiber lobe neurons

Voltage-dependent ionic currents were recorded from squid giant fiber lobe neurons using the whole-cell patch-clamp technique. When applied to the bathing solution, methadone was found to block IK, I Na and I Ca. Both I Na and I Ca were reduced without apparent change in kinetics and exhibited IC(50)'s of 50-100 and 250-500 mu M, respectively, at +10 mV. In contrast, IK was reduced in a time-dependent manner that is well fit by a simple model of open channel block (K(D)= 32+/- or 2 mu M, +60 mV, 10 degrees Celsius). The mechanism of I(K) block was examined in detail and involves a direct action of methadone, a tertiary amine, on K channels rather than an opioid receptor-mediated pathway. The kinetics of I(K) block resemble those reported for internally applied long chain quaternary ammonium (QA) compounds; and recovery from I(K) block is QA-like in its slow time course and strong dependence on holding potential. A quaternary derivative of methadone (N-methyl- methadone) only reproduced the effects of methadone on I(K) when included in the pipette solution; this compound was without effect when applied externally. I(K) block thus appears to involve diffusion of methadone into the cytoplasm and occlusion of the open K channel at the internal QA blocking site by the protonated form of the drug. This proposed mode of action is supported by the pH and voltage dependence of block as well as by the observation that high external K+ speeds the rate of drug dissociation. In addition, the effect of methadone on I(K) evoked during prolonged (300 ms) depolarizations suggests that methadone block may interfere with endogenous K+ channel inactivation. The effects of temperature, methadone stereoisomers, and the methadone- like drugs propoxyphene and nor-propoxyphene on IK block were examined. Methadone was also found to block I(K) in GH3 cells and in chick myoblasts.     

Open channel noise. IV. Estimation of rapid kinetics of formamide block in gramicidin A channels.

Blocking events in currents through biological ion channels occur over a wide range of characteristic times. The interruptions in single-channel currents from blocking events may be characterized by the direct measurement of gap durations or by analyzing open-channel current histograms, provided that the events are not much shorter than the time resolution of single-channel recordings (approximately 10 microseconds). Here we present a method for the characterization of channel block on a much faster time scale by combining open-channel noise measurements with subsequent model fits according to a theoretical approach (Frehland, E. 1978. Biophysical Chemistry. 8:255-265). Although the bandwidth limitations in open-channel noise experiments are the same as in conventional single-channel experiments, from the dependence of the mean current and the spectral density of the noise on the concentration of the blocking agent, kinetics of very brief blocking events can be estimated. As an example we have analyzed the open-channel noise of K+ currents through the gramicidin A channel in the presence of various concentrations of formamide, a weak blocker, at neutral pH. We estimate the blocking and unblocking rates to be approximately 10(7)s-1 at 1 M formamide and discuss possible mechanisms for the blocking process.     

Channel blocking properties of a series of nicotinic cholinergic agonists.

Inhibition of the nicotinic acetylcholine receptor (nAChR) by channel blockade has been demonstrated with a variety of large organic cations, including several nicotinic agonists. We have studied the kinetics of channel blocking of a series of agonists which vary systematically in size and hydrophobicity due to a hydrocarbon chain from one to six carbons in length, as well as one agonist with a tertiary isomer of one hydrocarbon chain. Single-channel recording was used in combination with three different analysis techniques for determining the kinetic and equilibrium parameters of channel blockade. With an increasing number of methylenes, the blocking rates were essentially constant and the unblocking rates decreased exponentially. This is consistent with studies of the blocking properties of alcohols at the nAChR channel. Also, a linear decrease in the depth to which the larger agonists penetrate the membrane spanning region of the channel was observed. The three smaller agonists, however, all traverse approximately 75% of the membrane field, in agreement with previous measurements of the location of the narrowest region of the channel, the selectivity filter.