Correspondence to: Edward Moczydlowski, Department of Pharmacology, Yale University Medical School, 333 Cedar St., New Haven, CT 06520-8066. Fax:203-785-7670 E-mail:edward.moczydlowski{at}yale.edu.
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Abstract |
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Many large organic cations are potent blockers of K+ channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na+ channels is an important determinant of Na+/K+ discrimination, exclusion of Ca2+, and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA+) that block Na+ channels in a voltage-dependent fashion. We investigated block by a series of TAA+ cations of the wild-type rat muscle Na+ channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA+ and larger TAA+ cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA+ cations as revealed by a positive inflection in the macroscopic IV curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA+) symmetrical TAA+ cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA+ cations with a molecular diameter as large as 15 Å can permeate Na+ channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na+ channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA+ cations and potentially other kinds of local anesthetic molecules.
Key Words: local anesthetic, ionic selectivity, Na+ channel, selectivity filter, tetraethylammonium
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INTRODUCTION |
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Tetra-alkylammonium (TAA+)1 cations such as TEA+ are known to inhibit ionic currents through channel proteins by transiently binding in the ion conduction pathway and blocking the flow of current (
Voltage-gated Na+ and Ca2+ channels are structurally related to K+ channels as members of a homologous superfamily of cation-selective channels (
The enhanced permeability of the DEAA mutant to large organic cations provides a unique opportunity to explore the mechanism of block by TAA+ cations and related local anesthetic drugs that are known to preferentially block voltage-gated Na+ channels from the intracellular side. If the K(III) residue of the DEKA locus corresponds to the location of a major energy barrier for the movement of large cations through the pore, then lowering this energy barrier by mutation may enhance the permeability of blocking cations that enter the channel from the inside. Alternatively, if other significant energy barriers are located between the DEKA locus and the intracellular pore entrance, then such mutant Na+ channels may exhibit asymmetric permeability to organic cations. On this basis, we hypothesized that removal of a structural element such as a Lys residue that limits conduction of organic cations may reveal or enhance a phenomenon known as voltage-dependent relief of block (
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MATERIALS AND METHODS |
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Expression of Wild-Type and Mutant Na+ Channels
Na+ channels studied in this paper are the wild-type µ1 rat skeletal muscle isoform (
Solutions and Electrophysiology
The standard extracellular Na+ bath solution was (mM) 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES-NaOH, pH 7.3. The standard intracellular Cs+/Na+ pipette solution was (mM) 125 CsF, 2 MgCl2, 1.1 EGTA, 10 glucose, 20 Na+-HEPES, pH 7.3. Intracellular pipette solutions containing tetrapropylammonium (TPA+), tetrabutylammonium (TBA+), TPeA+, THexA+, and QX-314+ were prepared by adding these blockers to standard Cs+/Na+ pipette solution at the desired concentration. The composition of other solutions for testing permeability to external TEA+, TMA+, MA+, and Ca2+ or block by internal TEA+ are given in the figure legends.
Patch-clamp electrodes were constructed with a commercial pipette puller using Kimax 50 borosilicate capillary glass (Fisher Scientific Co.) without additional fire polishing. The measured pipette resistance was 12 M when filled with standard Cs+/Na+ pipette solution. Whole-cell voltage-clamp recording was performed at room temperature (~22°C) using an amplifier (EPC-9; HEKA Electronik) with Pulse and Pulse-fit software (Instrutech Corp.). Stably transfected HEK293 cells were seeded for growth on glass cover slips and used for electrophysiological recording within 1236 h. Transiently transfected cells expressing the DERA mutant channel were used for recording 48 h after transfection. The peak inward current in standard Na+ bath solution and Cs+/Na+ pipette solution of typical cells selected for recording DEKA, DEAA, and DERA current was -4.3 ± 0.4 nA (mean ± SEM, n = 6), -4.1 ± 0.7 nA (n = 12), and -1.6 ± 0.4 nA (n = 5), respectively. Cancellation of residual capacitance transients and linear leak subtraction was carried out using a programmed P/4 negative pulse protocol delivered at -120 mV. The series resistance compensation function of the amplifier was routinely used at 7479% compensation to minimize voltage error. Nevertheless, some experiments for the DEAA mutant (e.g., see Fig 2 and Fig 5) involved measurements of outward currents as large as 50100 nA at the highest positive voltage (+200 mV). In such cases, the effect of voltage errors on our analysis and interpretation was minimized by discarding data sets from cells that exhibited >50 nA of outward current at +200 mV. In addition, the effect of voltage error due to series resistance, RS, was estimated for the data of Fig 2 and Fig 5 by correcting the applied voltage by the voltage drop across the noncompensated fraction, fN, of measured RS (2.3 ± 0.2 M
, n = 5), using Ohm's Law,
V = IfNRS (
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Currentvoltage data were collected by recording responses to a consecutive series of step voltage pulses increasing by +10 mV from a holding potential of -120 to +200 mV with a pulse duration of 10 ms. The interval between consecutive voltage pulses was 0.5 or 1 s as noted, except in experiments where it was varied from 0.5 to 10 s for investigation of use-dependent block. Data collection was begun 10 min after whole cell break-in when inward Na+ current reached a relatively stable level after intracellular perfusion. Data were collected during continuous gravity-fed perfusion of the extracellular bath solution using a commercial patch perfusion chamber (Warner Instrument Co.).
The permeability of extracellular TEA+ and MA+ was investigated by measuring the change in reversal potential, VR, upon changing the extracellular solution from the standard Na+ bath solution to a different solution containing the cation of interest (e.g., see Fig 1 A and 7). Permeability ratios of these extracellular cations relative to Na+ were calculated by the method of
VR values corrected for junction potentials and equations described previously (
Data Analysis and Modeling
Peak current values were measured and plotted as a function of the pulse voltage. In compiling the results, peak I-V data was often normalized by dividing the measured current by the maximum inward current of a given cell. Such data sets collected from three to eight cells were then averaged and plotted as the mean normalized current with error bars given (±SEM).
In Fig 2 B (below), current values for 10 and 50 µM internal TPeA+ were normalized by dividing each point by the expected peak inward current in the absence of internal TPeA+. The magnitude of the unblocked current was estimated by fitting data points at voltages less than +80 mV to a simple model of voltage-dependent block as described by Equation 1 and Equation 2 with outward permeation of the blocker disregarded by setting k-2 equal to 0. This procedure yields a reasonable estimate of the maximal conductance, Gmax, in the absence of blocker. This estimate of Gmax is then used to generate an I-V relation for the expected unblocked current by setting the blocker concentration, [Bin], equal to zero in Equation 1. The absolute value of the maximal expected inward current is then used to normalize the measured current in the presence of internal TPeA+ to produce a macroscopic I-V curve that is appropriately scaled with respect to that expected in the absence of an internal blocker. This procedure is mathematically similar to that used in Figure 5 of
Nonlinear regression fitting of peak I-V data to the model of Scheme 1 using Equation 1 and Equation 2 was performed using the Marquardt-Levenberg algorithm as part of the Sigmaplot 4.0 software package (SPSS Inc.).
Materials
Chloride salts of TAA+ cations were purchased from Aldrich Chemical Co. (TPA+, TBA+, TPeA+) or Sigma Chemical Co. (TEA+, THexA+). Saxitoxin and tetrodotoxin were purchased from Calbiochem Corp. µ-Conotoxin GIIIB was obtained from Bachem. The quaternary lidocaine derivative QX-314+ was obtained from Alomone Laboratories.
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RESULTS |
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Voltage-gated Na+ channels are preferentially blocked by many different hydrophobic organic cations from the intracellular side. For example, QX-314+, the quaternary ammonium derivative of lidocaine, a local anesthetic, readily blocks Na+ channels when present on the intracellular side of axons, cells, or planar bilayers (
Asymmetry of Organic Cation Permeation through the DEAA Mutant Na+ Channel
The control experiment of Fig 1 shows that replacement of a standard extracellular Na+ solution with a solution containing TEA+ as the major external cation results in a small inward current and a negative shift of the reversal potential of whole-cell peak current for the DEAA mutant. The magnitude of this shift (-24.6 ± 0.3 mV) corresponds to a permeability ratio of PTEA(out)/PNa = 0.37 ± 0.01 (n = 4), confirming that external TEA+ can permeate through this channel. TEA+ is the largest cation that has thus far been found to exhibit measurable inward current for the DEAA mutant (
Relief of Block by Internal TPeA+ at High Positive Voltage in DEAA Mutant and Wild-Type Na+ Channels
The next question we addressed was whether block of outward alkali cation current by large hydrophobic TAA+s is altered in the DEAA mutant compared with wild type. Internal TPeA+ has been previously characterized as a potent blocker of outward Na+ current for the wild-type human heart Na+ channel (
However, at voltages greater than +140 mV, there is a sharp upturn of the peak I-V relationship with 10 and 50 µM TPeA+ (Fig 2 B). Inspection of the corresponding current traces (Fig 2 A) shows that outward currents recorded with 10 and 50 µM TPeA+ display typical rapid activation and inactivation kinetics of voltage-gated Na+ channels. A positive inflection in the peak I-V relations of Fig 2 B arises from a rather abrupt increase in transient current at voltages greater than +140 mV (Fig 2 A). This kind of behavior is consistent with voltage-dependent relief of TPeA+-blocked Na+ channels, rather than an artifact due to activation of some other type of channel with different kinetics in HEK293 cells. Visual comparison of the time course of the outward current in the absence and presence of TPeA+ shows that the apparent rate of inactivation is distinctly faster with TPeA+ (Fig 2 A). This phenomenon was also observed by
Because the absolute outward current in HEK293 cells expressing the DEAA mutant can be as large as 100 nA, we considered whether residual series resistance error might lead to distortions in the measured peak I-V relations that could affect our interpretation. Uncompensated series resistance leads to distortions in the current time course and a failure to maintain the desired clamp voltage (
An inconvenient aspect of quantitative analysis of internal blockers by whole-cell patch clamp is that the internal solution cannot be readily changed, so that the level of unblocked control current for a given cell with internal blocker is difficult to establish. However, since TAA+ cations are well behaved voltage-dependent blockers of Na+ channels in the low voltage range of +80 mV or less ( 13.2 Å) has not been previously reported for a cation-selective channel that has a rather narrow selectivity filter, on the order of 3 x 5 Å for the wild-type Na+ channel (
One mechanism by which large molecules might pass through a small pore could involve physical deformation or a transient enlargement of the limiting region of the filter. To investigate this possibility, we tested the sensitivity of the relief-of-block phenomenon to toxins that block the Na+ channel from the external side. The experiments of Fig 3 show that outward currents associated with relief of block by 50 µM internal TPeA+ are partially inhibited by 40 µM STX and 1 µM µ-conotoxin GIIIB (µ-CTX). When bound to the Na+ channel, these particular toxins interact with numerous residues in the outer mouth that are thought to be located near the entrance to the selectivity filter (30 min during continuous perfusion of the extracellular solution and multiple rounds of stimulation at high positive voltage. (As a finer point of interpretation, it is worth noting that the data of Fig 3B and Fig C, suggest that STX and µ-CTX appear to be more effective at inhibiting inward current than outward current in the presence of internal TPeA+. This may be due to trans effects of current flow or transient changes in toxin affinity associated with block or relief-of-block that will require further study to fully characterize.)
In studying relief of block by TPeA+ in the DEAA mutant, we first hypothesized that voltage-driven permeation of this cation must be specifically related to a significant increase in the cutoff diameter of this mutant for organic cation permeation as previously found in molecular sieving studies (
The Relief-of-Block Phenomenon Is Enhanced for Long Chain TAA+ Derivatives
The next question we addressed concerned the structural requirements of organic cations that favor the relief-of-block phenomenon. We tested the relative ability of the series of symmetrical TAA+ cations to exhibit relief of block as judged by a positive inflection in the whole-cell peak I-V curve taken with various blockers in the pipette solution. Blockers ranging in size from TEA+ to TPeA+ were tested at concentrations at or above their reported blocking Kd at 0 mV for native heart Na+ channels (
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CurrentVoltage Behavior in the Presence of Internal TPeA+ Depends on the Frequency of Stimulation
The binding of many local anesthetic drugs to an internal blocking site of voltage-gated Na+ channels is known to exhibit use dependence, a phenomenon in which the occupancy of the site by the drug increases with the frequency of stimulation (
Use-dependent block of Na+ channels is traditionally monitored by a series of identical depolarizing voltage pulses delivered at various frequencies of repetitive stimulation. However, in the present case, we were interested in whether use dependence affects the shape of the peak I-V curve collected here by a series of increasingly positive voltage steps. To examine this possibility, we varied the rate at which consecutive points of the peak I-V relation are collected. The standard protocol for taking I-V data in this study used a waiting period of 0.5 s between consecutive voltage pulses, starting from -140 to +200 mV. This corresponds to a stimulation frequency of 2 Hz between successive I-V data points. Fig 6 B shows that the shape of the peak I-V relation for outward current of the DEAA mutant exposed to 50 µM internal TPeA+ depends on the rate of consecutive voltage pulses. At a low stimulation rate of one pulse every 10 s (0.1 Hz), the positive inflection of the outward I-V relation is rather shallow. As the stimulation rate is increased from 0.1 to 2 Hz, the plateau region of the I-V curve observed in the range of +50 to +150 mV is enhanced. This effect corresponds to stronger block that would be expected if a higher stimulation rate increases the occupancy of the blocking site by TPeA+. At all tested frequencies, a positive inflection in the I-V curve is still observed in the high voltage range, but the transition from blocking behavior to relief-of-block is accentuated at higher stimulation frequency. This is consistent with the idea that the positive inflection in the I-V curve represents voltage-dependent exit of TPeA+ from the blocked/inactivated state of the Na+ channel that also occurs in the phenomenon of use-dependent block. Fig 6 A shows that the wild-type Na+ channel responds in a similar fashion to increasing stimulation frequency in this experiment; however, the relative enhancement of block in the positive voltage range is much less than that of the DEAA mutant. This implies that the blocking kinetics of TPeA+ must differ for the wild-type versus mutant channel.
The DERA Mutant also Exhibits Relief of Block by TPeA+
To further investigate how the amino acid residue at the K(III) position of the DEKA locus affects the relief-of-block phenomenon, we examined this behavior in a DERA mutant in which the Lys residue at this position is replaced by Arg. We previously showed that the DERA mutant of the µ1 Na+ channel is nonselective toward Na+ versus K+ and does not support a macroscopically detectable inward Ca2+ current in Xenopus oocytes (
We first confirmed that the DERA mutant is impermeable to external Ca2+ when expressed in HEK293 cells. This is shown by the experiment of Fig 7 A in which replacement of extracellular Na+ solution by Ca2+ solution results in a complete loss of inward current, a result that is quite similar to that obtained for the DERA mutant expressed in Xenopus oocytes (
We next tested whether the molecular sieving behavior of the DERA mutant is altered with respect to external organic cations. The wild-type µ1 Na+ channel is impermeant to external MA+, whereas this organic cation supports a sizeable inward current through the DEAA mutant that is 32% of the peak inward Na+ current with a calculated permeability ratio of PMA/PNa = 0.41 ± 0.04 (
Fig 8 A shows the whole-cell peak I-V behavior up to +200 mV of the DERA mutant with 125 mM Cs+ plus 20 mM Na+ as the major internal cations. This mutant exhibits a much larger relative outward current than the wild-type DEKA channel under these conditions (compare Fig 8 A and 5 A). However, the corresponding relative outward conductance of the DEAA mutant is still about twice as large and more ohmic in nature than that of DERA (compare Fig 2 B and 8 A). Therefore, the amino acid residue at the K(III) position of the DEKA locus seems to be an important determinant of I-V rectification behavior of outward current through the µ1 Na+ channel. The peak I-V relation of the DERA mutant also exhibits a reproducible positive inflection in the vicinity of +140 mV in the absence of any added internal blockers (Fig 8 A). We have not yet determined the basis of this interesting behavior, but we offer two possible explanations that require further investigation: (a) the Arg residue at the K(III) position of the DEKA locus may introduce or alter some kind of intrinsic rectification of the Na+ channel pore with respect to alkali cations, and (b) the Lys(III)/Arg(III) residue controls the permeability of an endogenous blocking molecule.
The peak I-V behavior of the DERA mutant with 10 µM TPeA+ in the internal solution is shown in Fig 8 B. This data indicates that internal TPeA+ blocks the DERA mutant in a use-dependent fashion as demonstrated by a marked enhancement of a plateau phase of the I-V curve in the range of +50 to +140 mV by increasing frequency of stimulation from 0.1 to 2 Hz. This behavior is rather similar to that of DEAA channel (compare Fig 8 B and 6 B). There is also a sharp upward inflection of the I-V curve for DERA in the presence of internal 10 µM TPeA+ at high positive voltage and at high stimulation frequency (Fig 8 B). The lack of similar use dependence for the endogenous I-V inflection in the absence of internal TPeA+ (Fig 8 A) distinguishes this phenomenon from the TPeA+ blocking effect and indicates that the DERA mutant supports significant relief of block by TPeA+. Thus, the presence of an Arg residue at the K(III) position of the DEKA locus does not seem to seriously impede the ability of TPeA+ to be driven outward through the selectivity filter at high voltage.
Relief-of-Block by TAA+ Cations Can Be Simulated by a Simple Kinetic Model of a Permeant Blocker
To assess whether I-V behavior in the presence of large TAA+ cations in the internal solution can be described by a mechanism based on voltage-dependent relief of block, we used a simple kinetic model to simulate this behavior for the DEAA mutant. This model is similar to one previously used by
In Scheme 1, an internal blocker, Bin, reversibly binds to an internally accessible site in the open channel, O, with an equilibrium dissociation constant of K1 = k-1/k1. Binding of Bin results in a nonconducting blocked state, OB. Outward permeation of the blocker is governed by the k-2 rate constant, which leads to recovery of the open state and infinite dilution of the blocker in the external solution. Since outward current of the DEAA mutant exhibits nearly ohmic behavior in the absence of internal blocker, an expression for simulating peak whole-cell current for this channel as a function of voltage can be approximated by an ohmic conductance multiplied by the probability that the channel is opened by activation gating and the probability that the channel is not occupied by the internal blocker. Ohm's law can be used to describe channel conductance and a standard Boltzmann expression can be used to simulate the probability of channel activation as a function of voltage. The probability that the channel is not blocked can be computed by using the apparent blocker dissociation constant for Scheme 1: KB = (k-1 + k-2)/k1. This relationship can be written as:
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(1) |
where Gmax is the maximal conductance, VR is the reversal potential, q is an effective gating charge for voltage activation, V0.5 is the midpoint voltage of activation gating, A is a constant equal to RT/F, or 25.4 mV, and [Bin] is the internal blocker concentration. It can be shown that KB(V) for Scheme 1 is equivalent to the product of the voltage-dependent equilibrium dissociation constant for internal block in the absence of an external permeation path, K1(V), times a term that contains the quantity, R(V) = k-2(V)/k-1(V), which is the ratio of the rate constant of blocker dissociation to the external solution over that of the internal solution. These relationships and their voltage dependence can be expressed as:
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(2) |
In Equation 2, K1(0), k-2(0), and k-1(0) are the values of the respective equilibrium and rate constants at 0 mV, and z is the valence of the blocker (+1 for TAA+). 1 is the summed fraction of electrical distance sensed by the blocker for the k1 step plus the k-1 step, and
2 is the summed fraction of electrical distance attributed to the k-2 step plus the k-1 step. The latter use of summed electrical distances in this simplified model is used to reduce the number of free parameters for the purpose of data fitting.
By empirical testing, we found that parameters for fitting this model to the actual macroscopic I-V data are closely constrained since Gmax, VR, q, and V0.5 are well defined by data points in the negative to low positive range, where blocking phenomena have only a small contribution. The behavior in the high positive voltage range is governed by KB(0) and 1, which determine current inhibition (block). Relief of inhibition at high voltage is governed by R(0), the ratio of k-2(V)/k-1(V) at 0 mV, and
2, the lumped electrical distance of the k-2 step plus the k-1 step. Fig 9 shows that this model can readily simulate the unusual shape of macroscopic peak I-V curves of the DEAA channel in the presence of TBA+, TPeA+, and THexA+. The average blocking parameters for fitting these data are summarized in Table 1. The fitted values of K1(0) and
1 for TBA+ and TPeA+ are similar to those reported for internal block of the human heart Na+ channel by these compounds in the voltage range less than +80 mV (
1 = 0.46 for TBA+ and 0.41 for TPeA+. The fitted values of R(0) listed in Table 1 indicate that the k-2 rate of outward permeation for TBA+ and TPeA+ only has to be ~23 x 10-4 of the k-1 rate for dissociation of these blockers back to the inside compartment to simulate the kind of blocking relief observed in this system. Table 1 also shows that the enhanced relief of block observed for THexA+ can be explained by a 100-fold increase in the value of R(0) for this particular molecule. This implies that THexA+ is able to exit through the channel to the outside much more readily that TBA+ and TPeA+. Table 1 also shows that large values of
2
1.0 are required to simulate the steep voltage dependence of the relief of block observed at high positive voltage. This indicates that additional charge-dependent processes besides the simple interaction of a single cation with a site in the transmembrane electric field are involved in generating this phenomenon.
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We must also caution that these simulations have certain quantitative limitations. The model assumes that the measured peak Na+ currents are proportional to the steady state interaction of the blockers with the open channel. This does not take into account the shortening of the apparent rate of channel inactivation that is evident in the current traces with large TAA+ cations. This effect is especially dramatic at high positive voltage for current records in the presence of TBA+ shown in Fig 9. The simulations also do not consider the use or frequency dependence inherent in collecting the I-V data of Fig 9. Despite these deficiencies, the modeling exercise shows that I-V data for the DEAA mutant Na+ channel are closely mimicked by a simple theory of voltage-dependent relief of block. Corrections to the model to account for kinetic effects will change absolute estimates of the blocking parameters, but will not affect the central conclusion that this process represents voltage-driven permeation of large organic cations.
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DISCUSSION |
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The primary goal of this study was to determine whether the K(III) residue of the Na+ channel DEKA locus plays a role in controlling intracellular blocking interactions of TAA+s. In the course of this investigation, we found that the rectification behavior of outward current is strongly affected by two different mutations of the K(III) residue. We also observed an unexpected phenomenon involving voltage-dependent relief of block by large TAA+ cations.
The first conclusion is that substitution of a butylammonium group with a methyl group that is effectively achieved by mutation of Lys(III) to Ala(III) in the DEAA mutant increases the cutoff diameter for molecular sieving ( in the range 0.40.7 based on the
An interesting new feature of the DEAA channel described here is that whole-cell current carried by internal alkali cations (Cs+, Na+) in the absence of other added blockers is nearly ohmic at positive voltages up to +200 mV (e.g., Fig 5 B). This stands in strong contrast to inwardly rectifying or sublinear I-V behavior of the wild-type DEKA channel in this voltage range (e.g., Fig 5 A) recorded under the same conditions with Na+ outside and Cs+ inside. This feature of the DEAA mutant makes it easier to recognize the dramatic relief of block by large TAA+ cations at voltages greater than +140 mV (e.g., Fig 2 B). The wild-type DEKA channel also apparently exhibits a similar phenomenon (Fig 4), but it is more difficult to analyze because of the background inward rectification. In this respect, a second conclusion of our study is that the K(III) residue of the DEKA locus controls the rectification behavior of outward macroscopic current of the µ1 Na+ channel expressed in HEK293 cells. The outward I-V relation of the DERA mutant with the Lys(III) to Arg substitution is more linear than the wild-type DEKA channel, but a distinctive negative inflection in the I-V curve of the DERA mutant seen with Cs+/Na+ pipette solution (Fig 8 A) further establishes that the particular residue at the K(III) position is a major determinant of current rectification.
The mechanism underlying the latter current inflection of the DERA channel and the sublinear I-V behavior of the wild-type channel is presently undetermined. However, our results provide clues that may help in its elucidation. Since block by various internal organic cations produces sublinear I-V behavior for the DEAA mutant, and certain mutations of the K(III) residue enhance permeation of organic cations, an obvious possibility is that an endogenous internal blocking molecule is partially responsible for inward rectification of the wild-type channel. As mentioned in RESULTS, our initial experiments suggest that Mg2+, Cs+, or HEPES, which are all present in the standard pipette solution, are not the only molecular species that may be responsible for this effect. At present, we suspect that other cellular cations such as polyamines may be involved in the endogenous rectification of wild-type Na+ channels, since polyamine block has been found to underlie inward rectification behavior of other classes of ion channels (
The third conclusion of this work is that voltage-dependent relief of block by the series of symmetrical TAA+ cations is facilitated by increasing length of n-alkyl chains. Where this phenomenon is easiest to study, in the DEAA mutant, there is little hint of relief of internal block due to TEA+, TPA+, or QX314+ by positive voltage up to +200 mV (Fig 5B and Fig C). In contrast, a distinct upturn in the I-V curve appears to commence at progressively lower voltages for the series, TBA+, TPeA+, and THexA+ (Fig 5 B and 9). As demonstrated by the modeling exercise of Fig 9, this behavior is consistent with voltage-driven permeation of large TAA+ cations to the outside of the channel. The structureactivity dependence of this effect implies that the permeation process is facilitated by hydrophobic interactions. However, this is a rather unusual type of hydrophobic interaction. The increase in hydrophobicity with increasing n-alkyl chain length of symmetrical TAA+ cations is accompanied by a proportional increase in molecular diameter, which would ordinarily be expected to inhibit permeation through a fixed-diameter pore in the usual manner of molecular sieving. Using energy-minimized molecular models of TAA+ cations (Hyperchem software from Hypercube), we find that the molecular diameter of these molecules increases by ~2 Å per symmetrical addition of a methylene group, as in the series: TMA+ (6.0 Å), TEA+ (8.2 Å), TPA+ (9.8 Å), TBA+ (11.6 Å), TPeA+ (13.2 Å), and THexA+ (15.2 Å), with the measured diameter given in parenthesis. The latter measurements are also in accord with an independent molecular dynamics analysis of the conformations and size of TAA+ cations in the absence of solvent (
Similar questions have been contemplated previously in the literature. In the case of inhibition of outward K+ current through squid axon delayed-rectifier K+ channels, block by both internal Na+ and Cs+ exhibits steeply voltage-dependent relief when external K+ concentration is low (
One mechanism that may account for the observed preference for large TAA+ molecules can be considered in reference to the molecular graphics illustration of Fig 10. For the sake of discussion, this figure shows space-filling models of the KcsA K+ channel, TEA+, and THexA+ at the same scale. The image on the left is a model of the KcsA structure with the four subunits alternately colored red and yellow. For orientation, Tyr82, a residue that constitutes an external aromatic binding site for TEA+ in related K+ channels (
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Although the illustration of Fig 10 is based on the crystal structure of a K+ channel protein, the pore domains of voltage-gated Na+ and Ca2+ channels are likely to have a similar structure since they belong to a superfamily of channel proteins consisting of evolutionarily related sequences ( subunit that contains four internally homologous domains, I, II, III, and IV. In any case, the molecular cutoff diameter of the selectivity filter of native Na+ channels (3 x 5 Å) is only slightly larger than that of K+ channels (3 Å). Fig 10 is thus a reasonable representation of the physical limitations that must underlie the improbability of electrodiffusion of large TAA+s through the Na+ channel pore. One implication of this subunit interface hypothesis is that K+ and Ca2+ channels may also exhibit relief of block by molecules such as TPeA+ at sufficiently high voltages. On the other hand, since the selectivity filter of Na+ and Ca2+ channels is based on the DEKA/EEEE signature motif of charged residues, specific structural features of these two channel subfamilies may be required for permeation of large TAA+s.
Another implication of this work is that the relief-of-block phenomenon ought to be useful for probing the mechanism of Na+ channel gating and the interactions of local anesthetic drugs. In the case of local anesthestics,
The notion that small molecules can enter cavities buried within the hydrophobic core of proteins by diffusing from the external solution is actually supported by a considerable body of evidence, mostly obtained from physical studies of soluble proteins. For example, fluorescence quenching and hydrogen exchange studies show that aromatic amino acid side chains and hydrogen atoms of peptide backbone amide groups buried in the interior of many proteins are accessible to collision and reaction with small molecules such as O2, I-, acrylamide, and H2O at relatively rapid rates of diffusion (
If this interpretation is correct, the relief-of-block phenomenon may provide a new tool to monitor and investigate the interactions of drug molecules with subunit interfaces of the Na+ channel. One approach would be to further examine the detailed structural requirements of organic cations for voltage-dependent relief of block. In this regard, it is interesting that QX-314+ does not exhibit significant relief of block in the assay described here (Fig 5B and Fig C). This molecule has been previously observed to block the cardiac isoform of Na+ channels from the outside of cells more readily than brain or muscle isoforms of Na+ channels (
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Footnotes |
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1 Abbreviations used in this paper: µ-CTX, µ-conotoxin GIIIB; MA+, methylammonium; STX, saxitoxin; TAA+, tetra-alkylammonium; TBA+, tetrabutylammonium; THexA+, tetrahexylammonium; TMA+, tetramethylammonium; TPA+, tetrapropylammonium; TPeA+, tetrapentylammonium; TTX, tetrodotoxin.
2 This estimate of 0.07 for PTMA(in)/PNa was calculated using the apparent reversal potential and an appropriate form of the Goldmann-Hodgkin-Katz voltage reversal equation for major monovalent cations. However, this calculation does not take into account the change in junction potential that occurs in whole-cell recording after diffusional equilibration between the pipette contents and cell interior. As described by
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Acknowledgements |
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This research was supported by a grant from the National Institute of General Medical Sciences of the National Institutes of Health (GM-51172 to E. Moczydlowski).
Submitted: 14 December 1999
Revised: 17 February 2000
Accepted: 22 February 2000
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