Skip Navigation

Cardiovascular Research 1999 42(2):503-509; doi:10.1016/S0008-6363(99)00024-3
© 1999 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Makielski, J. C
Right arrow Articles by Kyle, J. W
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Makielski, J. C
Right arrow Articles by Kyle, J. W
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1999, European Society of Cardiology

Intrinsic lidocaine affinity for Na channels expressed in Xenopus oocytes depends on {alpha} (hH1 vs. rSkM1) and β1 subunits

Jonathan C Makielski*, James Limberis1, Zheng Fan2 and John W Kyle1

Departments of Medicine and Physiology, University of Wisconsin, Madison, WI 53792, USA

jcm{at}medicine.wisc.edu

* Corresponding author. Present address: University of Wisconsin Clinics and Hospitals, 600 Highland Ave H6/349, Madison, WI 53792, USA. Tel.: +1-608-263-9648; fax: +1-608-263-0405

Received 30 September 1998; accepted 18 January 1999


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The affinity of lidocaine for the {alpha}-subunit of the Na channel has been reported to be greater for heart than for non-heart {alpha}-subunits, and also to be no different. Lidocaine block has a complex voltage dependence caused by a higher affinity for the inactivated state over the resting state. Inactivation kinetics, however, depend upon the {alpha}-subunit isoform and the presence of the auxiliary β1-subunit and will affect measures of block. Methods: We studied the voltage dependence of lidocaine block of Na currents by a two microelectrode voltage clamp in oocytes injected with RNA for the Na channel {alpha}-subunits of human heart (hH1a) or a rat skeletal muscle (rSkM1) alone, or coexpressed with the β1-subunit. Results: The midpoints of availability for a 25-s conditioning potential in control solutions were –65 mV for rSkM1, –50 for rSkM1+β1, –78 mV for hH1a and –76 for hH1a+β1. The Kd of tonic lidocaine block was measured at –90, –100, –110, –120 and –130 mV in the same oocytes. The apparent Kd for both isoforms ±β1 became greater with more negative holding potentials, but tended to reach different plateaus at –130 mV (Kd=2128 µM for rSkM1, 1760 µM for rSkM1+β1, 433 for hH1a, and 887 µM for hH1a+β1). Inactivated state affinities, assessed by fitting the shift in the Boltzmann midpoint of the availability relationship to the modulated receptor model, were 4 µM for rSkM1, 1 µM for rSkM1+β1, 7 µM for hH1a and 9 µM for hH1a+β1. Conclusion: The heart Na channel {alpha}-subunits expressed in oocytes have an intrinsically higher rest state affinity for lidocaine compared to rSkM1 after the voltage- and state dependence of block are considered. Coexpression with β1 modestly increased the rest affinity of lidocaine for rSkM1, but had the opposite effect for hH1a.

KEYWORDS Local anesthetics; Antiarrhythmic drugs; Sodium current; Modulated receptor model; Heart; Skeletal muscle; Ion channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Lidocaine is a clinically useful antiarrhythmic and local anesthetic that blocks the voltage-dependent Na channel. Lidocaine, and analogues such as the QX compounds, have also been used to probe Na-channel structure and function, as in, for example, locating the local anesthetic binding site to the inner pore (e.g., [1,2]). The affinity of lidocaine for the {alpha}-subunit of the Na channel has variously been reported to be greater for heart than for skeletal muscle [3–6] or to be no different [7–9]. At least part of the reason for these discrepant reports is probably caused by the dependence of lidocaine affinity on the kinetic state of the channel, as described in the modulated receptor model [10,11]. A predominant effect on lidocaine binding to Na channels is a dependence on the inactivated state. At any given rest or holding potential, the Na channel is distributed between resting and inactivated states, as described by the steady-state voltage-dependent availability from inactivation relationship or h{infty} curve. The h{infty} curve for non-heart isoforms, such as the skeletal muscle channel, is to the right (more depolarized) of that for heart; therefore, at any given potential, fewer channels are inactivated in skeletal muscle than they would be in heart. This results in less binding to the high affinity inactivated state and less block. The question then becomes, is the greater block demonstrated in heart caused by an intrinsic affinity difference for either the inactivated state or the resting state of the {alpha}-subunit, or can the apparent differences in affinity be accounted for by isoform differences in inactivation kinetics?

Most Na-channel function can be accounted for by expression of the pore-forming {alpha}-subunit alone, co-expression with the β1-subunit has been shown to affect inactivation kinetics (see [12,13] for reviews), and the β1-subunit decreases lidocaine affinity for the heart channel independently of the effect on kinetics [14]. The effect of the β1-subunit on lidocaine affinity in rSkM1 and any possible role for β1 in isoform differences in affinity has not been previously studied. This study was designed to investigate the voltage-dependence of apparent lidocaine binding affinities of the human heart channel {alpha}-subunit alone (hH1a), the heart {alpha}-subunit co-expressed with the β1-subunit (hH1a+β1), the rat skeletal muscle channel {alpha}-subunit alone (rSkM1), and the skeletal muscle {alpha}-subunit coexpressed with the β1-subunit (rSkM1+β1) with the aim of determining whether or not these subunits, either together or separately, have intrinsically different lidocaine affinities for the channel, or whether or not apparent differences might be accounted for by the different gating kinetics. Such differences may provide insights into channel structures responsible for drug binding. Some of these data have been presented previously in abstract form [15].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Na-channel {alpha}- and β1-subunit clones
The method of preparation of cRNA and Xenopus oocytes has been published previously [14]. The human heart Na-channel {alpha}-subunit (hH1a) was kindly provided by Drs. H. Hartmann and A.M. Brown, and the rat skeletal muscle channel (rSkM1) by Gail Mandel. The rat brain β1-subunit was cloned from rat brain poly(A)+ RNA as previously described [14].

2.2 Electrophysiological recordings in oocytes
Xenopus oocytes were injected with 50–150 ng of cRNA for hH1a alone, or in some cases, a mixture of cRNA for hH1a and cRNA for β1-subunits in three–nine-fold excess of that for hH1a or rSkM1 (total, 50–150 ng). One to seven days after injection, INa was recorded from cRNA-injected oocytes with a two electrode voltage clamp/bath clamp. A Dagan CA-1 (bath clamp) with a series resistance compensation circuit (TEV-208, Dagan) was used to make recordings. All recordings were made at 20–22°C in a flowing bath solution consisting of (in mM): 96 NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2 and 5 Hepes, pH 7.2. Lidocaine in appropriate amounts was added from a 100-mM stock solution in distilled water. Electrodes contained 3 M KCl and had resistances that ranged from 0.2 to 1.5 M{Omega}. Data were acquired using pClamp6 software (Axon Instruments) using an Intel-486-based computer. Data were digitized at 42 kHz and were low pass filtered at 10 kHz (–3dB).

2.3 Voltage control and preparation stability considerations in whole oocytes
Whole oocyte preparations offer stable recordings of INa kinetics for up to 4 h or more, allowing for more prolonged protocols and study of multiple drug concentrations including a washout of drug. Although not done in all oocytes for which data are reported, a washout of drug could be uniformly achieved . Oocytes were generally studied on the second day after injection for rSkM1 and the third day after injection for hH1a. Peak current for a depolarization to –10 mV from a holding potential of –120 mV were –3.74±2.3 µA (n=8) for hH1a, –2.99±2.3 µA (n=6) for hH1a+β1, –3.53±2.09 µA (n=9) for rSkM1 and –2.56±1.59 µA (n=8) for rSkM1+β1. Over several hours, the oocytes sometimes exhibited current ‘run-up’, or a gradual increase in current density. Run-up presumably occurred at times as a result of continuing channel expression during the course of the experiment. Normalization for run-up is described in the analysis section. The large membrane area of the oocyte (about 1 mm diameter spheres; Cm>200 nF) prevents the membrane potential from rising fast enough following step changes in the whole oocyte clamp to accurately study activation Na currents. The intracellular potential monitored by the third electrode achieved the target potential after the decay of the capacity surge and before peak Na current (Fig. 1). For all protocols, peak current was used as an assay to assess Na-channel availability and gave results similar to those obtained with macropatch, where charging times are less of a problem [14]. Voltage control problems caused by current flow over series resistance during INa was not a problem, as Vm was monitored during most of the experiments and deviations from command were less than 1 mV.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 INa traces in control (solid lines) and in 500 µM lidocaine (dotted line) for the heart {alpha}-subunit isoform alone (hH1 top, left), skeletal muscle isoform {alpha}-subunit alone (rSkM1, top right), hH1 co-expressed with the β1-subunit (bottom, left) and rSkM1 coexpressed with the β1-subunit. Currents were elicited by a depolarization from a holding potential of –120 mV to –10 mV. Calibration bars represent 0.5 µA and 1.0 ms. Tonic block of peak current was assessed by taking the difference between the peak current in control and in lidocaine and dividing by the peak current in controls. In these examples, tonic block was 50% for hH1a, 39% for hH1a+β1, 22% for rSkM1 and 43% for rSkM1+β1.

 
2.4 Experimental protocols and analysis
Details of the voltage protocols are provided with the data. The standard holding potential for whole oocyte clamp was –120 mV. Peak currents were measured as the maximum negative current after the depolarization. Leak correction was unnecessary because the expressed currents were always more than 20-fold the amplitude of endogenous oocyte currents and passive leak. Residual outward capacity current at the time of peak current, however, caused an underestimation of peak INa for uncorrected traces. To correct for this, a capacity transient from a steady-state inactivation protocol for which all INa was inactivated (conditioning potential>–20 mV) was used to correct peak currents for the standard test protocol of –10 mV. This capacity transient was for a 110-mV step (from –120 to –10 mV) in both cases because the protocol contained a 2-ms step to –120 mV between the conditioning and the test potential. The frequency of stimulation within a protocol was carefully considered and tested so that no effects of a previous stimulation would be present in a subsequent test. This often required intervals of many minutes between single test pulses in a protocol and experimental durations extending for many hours. The run-up phenomenon described earlier, if uncorrected, affected measures of tonic block at different potentials and drug concentrations done at later times. To account for this, INa at –120 mV in control (IC) and immediately after equilibration with lidocaine (IL) was measured, an interval in which run-up is negligible. Corrected control currents for –120 mV (ICC–120) were calculated from subsequent measurements of current in lidocaine (ISL–120) by a simple ratio ICC–120=ISL–120IC/IL. The effect of this correction process was to normalize the relative tonic block for different potentials to that at –120 mV, where reliable absolute measures of tonic block had been obtained. Data were fit to model equations using non-linear regression (Procedure NLIN) using SAS (Cary, NC) statistical software running on a SUN IPX workstation. Parameter estimates are given ± the standard error for the parameter estimate.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Fig. 1 shows examples of INa recorded for depolarizations to –10 mV from –120 mV for four different oocytes expressing hH1a±β1 and rSkM1±β1. As reported previously (see [12,13] for review), the decay of INa for the rSkM1 {alpha}-subunit alone was slow but was more rapid when expressed with the β1-subunit, whereas the decay for hH1a was rapid and not affected by co-expression with the β1-subunit. Also shown (dotted lines) are INa traces recorded 5–10 min after adding 500 µM lidocaine to the bath solution. These raw data examples suggest that the efficacy sequence for tonic lidocaine block at –120 mV is hH1a>hH1a+β1>rSkM1+β1>rSkM1. Lidocaine block, however, is very sensitive to the holding potential caused by the greater affinity of lidocaine for the inactivated state over the resting state of the channel.

The voltage-dependent availability from inactivation relationship (h{infty}) was determined by applying a 25-s-long conditioning step to various potentials followed by a test depolarization to –10 mV, to assess the availability of channels to open. This relatively long conditioning step was used because we had determined previously by applying conditioning potential of various durations that parameters of the h{infty} relationship were not near steady-state for conditioning potential durations <10 s for hH1a±β1 [14], and we also confirmed that this was the case for rSkM1±β1 (data not shown). Fig. 2 shows examples of h{infty} relationships for the two {alpha} isoforms ±β1 in control solutions (filled symbols). These data show that the midpoints (V1/2) of h{infty} from most negative to positive are hH1a<hH1a+β1<rSkM1<<rSkM1+β1. Summary data for the midpoints are given in Table 1. Fig. 2 also shows examples of the effect of 500 µM lidocaine on the h{infty} relationship to cause a shift in apparent h{infty} in the hyperpolarizing direction (arrows). This shift is interpreted below in terms of high affinity inactivated state block.


Figure 2
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Examples of steady-state availability of INa for hH1a (top) and rSkM1 (bottom) with and without coexpression of the β1-subunit and in control and in the presence of 500 µM lidocaine. The data points were obtained by measuring peak currents in response to a test depolarization to -10 mV following a 25-s conditioning depolarization to the various conditioning potentials (Vc) plotted on the abscissa. A 2-ms interval at –120 mV was interposed between the conditioning and test steps in order to normalize the capacity transients. Peak INa were fit to a Boltzmann equation: Peak INa=INa–max[1+exp(Vc–V1/2)/{kappa}]–1, where INa–max is the predicted maximum peak INa, V1/2 and {kappa} are the mid-point and slope, respectively, of the Boltzmann relationship. The solid lines represent fits of non-normalized data to the Boltzmann equation, which were later normalized by dividing by the fitted parameter INa–max. For plotting data, peak INa versus Vc peak currents were normalized to the largest peak obtained. Top panel: For hH1a without β1 (bullet), V1/2=–81.4±0.1 mV and {kappa}=5.0±0.1; the addition of lidocaine [500 µM ({circ})] caused a 20.1 mV shift (-> on graph) in the midpoint to V1/2=–101.5 mV±0.3 and {kappa}=5.9±0.2. When hH1a was co-expressed with the β1-subunit ({blacktriangledown}), the midpoint was more positive by 5 mV, i.e. V1/2=-74.2±0.2 mV and {kappa}=5.3±0.2 than without β1, and the addition of 500 µM lidocaine ({triangledown}) shifted (-> on graph) the midpoint by 23.5 mV to V1/2=94.7±0.2 mV, {kappa}=5.4±0.2. Bottom panel: for rSkM1 without β1 (bullet), V1/2=–68.1±0.1 mV and {kappa}=5.0±0.1; the addition of lidocaine [500 µM ({circ})] caused a –17.4 mV shift (-> on graph) in the midpoint to V1/2=–85.5 mV±0.2 and {kappa}=5.4±0.1. When rSkM1 was co-expressed with the β1-subunit ({blacktriangledown}), the midpoint was more positive by 23.7 mV, i.e. V1/2=–44.2±0.9 mV, {kappa}=6.1±0.7, than without β1, and the addition of 500 µM lidocaine ({triangledown}) shifted (-> on graph) the midpoint by 27.8 mV to V1/2=72.2±0.2 mV, {kappa}=5.4±0.2.

 

View this table:
[in this window]
[in a new window]

  		Table 1 State-dependent affinities and h{infty} parameter fits for {alpha}-subunit isoforms ±β1-subunita

 
To assess the effect of holding potential on the apparent dissociation constant for lidocaine from the channel (Kd), as measured by tonic block, we fit data for the decrease in current from control at various drug concentrations at –90, –100, –110, –120 and –130 mV for both isoforms with and without co-expression of the β1-subunit (Fig. 3). We plotted the apparent Kd for the {alpha}-subunits with and without the β1-subunit (Fig. 4). We were unable to study potentials negative to –130 mV because the preparation did not tolerate holding potentials negative to –130 mV for the required conditioning duration (25 s), but it is apparent from this figure that the values reached a plateau and that the value at –130 mV is likely to be a reasonable estimate of the dissociation constant for the resting state (Kdr); these values are reported in Table 1. We conclude that the heart {alpha}-subunit has a higher rest affinity for lidocaine than the skeletal muscle {alpha}-subunit. We had previously noted that co-expression of the β1-subunit decreased rest affinity of hH1a for lidocaine [14], and these new data confirm this observation. For rSkM1, however, the opposite effect is observed; co-expression with β1 appears to increase the rest affinity. The mechanism for these differing effects is not known.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Dose–response relationships for tonic block of peak INa by lidocaine at different holding potentials for the heart {alpha}-subunit isoform alone (hH1a top, left), skeletal muscle isoform {alpha}-subunit alone (rSkM1, top right), hH1a co-expressed with the β1-subunit (bottom, left) and rSkM1 coexpressed with the β1-subunit. Block was determined as in Fig. 1 from the peak INa in response to a test depolarization to –10 mV at the lidocaine concentration indicated on the abscissa after at least 25 s at holding potentials of –90 mV ({blacksquare}), –100 mV ({triangledown}), –110 mV ({blacktriangledown}), –120 mV ({circ}) and –130 mV (bullet). Data are shown as single data points for individual measurements and as means±standard deviation for between two and seven observations (total number of observations for each isoform are given in the table for Kdr). The lines represent fits to a single site binding relationship:

Figure 3
where Kd is the half blocking or dissociation constant for tonic block at each potential. The fit was made using non-linear regression to individual data points rather than the means, to allow for proper weighting of the data. The values for Kd are plotted against the holding potential at which they were determined in Fig. 4.

 

Figure 4
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Voltage dependence of the dissociation constant (Kd) for tonic block for the heart {alpha}-subunit isoform alone (hH1a, bullet), rat isoform {alpha}-subunit alone (rSkM1, {blacktriangledown}), hH1a co-expressed with the β1-subunit ({circ}) and rSkM1 coexpressed with the β1-subunit ({triangledown}). Kd was determined at different holding potentials from nonlinear regression fit to the data in Fig. 3. The symbols represent the parameter estimate, and the error bars are the standard error for the parameter estimate. Error bars are not shown in those cases where they are smaller than the symbols.

 
Lidocaine caused a negative shift in the h{infty} relationship (Fig. 2); this has been interpreted as being caused by increased binding to the inactivated state as the membrane is increasingly depolarized in the conditioning step, as proposed in the modulated receptor model [11] (see diagram inset to Fig. 5). Inactivated state affinity (Kdi) was estimated from the lidocaine concentration dependence of the shift in midpoint of the h{infty} relationship (Fig. 5) using the equations of Bean et al. [7] and the results are given in Table 1. These results suggests that rSkM1 had a greater inactivated state affinity for lidocaine than hH1a, and that rSkM1+β1 had a greater inactivated state affinity for lidocaine than rSkM1 alone, consistent with the data that show a greater shift in the midpoint for rSkM1+β1 than for rSkM1 alone, both of which were greater than for hH1a±β1. The fitting, as described in the figure legend, assumed a uniform slope of 5.0 and the resting affinities, Kdr, as measured separately (Table 1) were explicitly stated. We also performed the fitting using other assumptions. Allowing Kdr to be fitted as a free parameter did not markedly change Kdi, but gave unrealistic values for Kdr (as measured more directly by separate protocols, see Figs. 1, 3 and 4GoGo). In general, however, Kdi estimates were not very sensitive to Kdr. When actual means of slopes were used (Table 1) rather than setting them to 5.0, the Kdi for hH1a±β1 changed minimally to 8 µM for hH1a and 9 µM for hH1a+β1, but the Kdi, for rSkM1 doubled to 2 µM for rSkM1 alone and 9 µM for rSkM1+β1. The estimated Kdi, therefore, are necessarily model-dependent and depend upon underlying assumptions, and the absolute values should be interpreted with caution. Lidocaine clearly, however, had a greater effect to shift the midpoint of the inactivation relationship for rSkM1 alone, as evident in Fig. 5, and this result is independent of modeling assumptions.


Figure 5
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 The shift in midpoint (V1/2) of the availability relationship with lidocaine was used to estimate the inactivated state affinity (Kdi). Availability was first determined in control solutions and then in the presence of lidocaine by the protocol described in Fig. 2, and the midpoint (V1/2) and Boltzmann slope were determined. The difference in the V1/2 between control and lidocaine is plotted against lidocaine concentration. The data are individual data points (n values are as given in Table 1). The lines are nonlinear fits of the data to the equation:

Figure 5
where Kdr is the affinity constant for the resting state of the channel and Kdi is the affinity for the inactivated state of the channel, and 5.0 is the approximate Boltzmann slope. See text for details of assumptions and alternative choices made in the fit. The fitted parameters±standard error of the parameter estimate are reported in Table 1.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Lidocaine exhibits the property of use-dependent block, that is, extra block accumulates with repetitive or prolonged depolarizations as a result of higher affinity binding to the inactivated state than for the resting state of the channel [10,11]. Lidocaine generally has a relatively stronger blocking effect on Na channels in native cardiac tissue over native nerve and skeletal muscle tissue. The relative sensitivity of lidocaine for Na channels in heart tissue may result from an intrinsic difference in lidocaine affinity for the different Na+-channel isoforms, but also could result from increased binding to the inactivated state in heart during the longer action potential, without any differences in affinity. In the first comprehensive voltage clamp study of lidocaine block on cardiac Na current, Bean et al. [7] compared their results with studies published previously for non-heart channels. They concluded that lidocaine showed similar interactions with the channel itself and attributed at least most of the difference to greater block in heart tissue during the greater dwell time in the inactivated state. One of the difficulties of using previously published data for comparison of heart with non-heart lidocaine binding, however, is that lidocaine block is sensitive to experimental conditions, including divalent ions, monovalent ions, pH, temperature, and to the specific voltage protocols used to elicit the block, in particular, holding potentials and conditioning potential durations. The advent of cloned isoforms of Na channels has allowed for the study of lidocaine block of these channels expressed in the same cell and under identical conditions.

4.1 Isoform differences in lidocaine affinity for the {alpha}-subunit
Affinity of lidocaine for the resting state of the channel is measured by holding the membrane at a potential that is sufficiently hyperpolarized that the majority of channels are in the resting state for a period of time that is sufficiently long for channels to enter the resting state and for equilibrium lidocaine binding to occur, then applying a test pulse and measuring the peak current as an index of the channels left unblocked. Previous reports in native [6] and in cloned channels [3–6] where lidocaine block was measured in heart and non-heart isoforms in the same study suggested that the resting state of the heart channel had an affinity that was two–four times greater than for non-heart isoforms. These studies, however, were all subject to the criticism that measurement of resting state affinity may have been contaminated by inactivated state block at the holding potentials used in the studies. At any given holding potential, a certain fraction of channels will be in the inactivated state. This fraction is much reduced at hyperpolarized potentials (as measured in the h{infty} relationship), but even very small fractions and differences in these fractions can affect block. According to the modulated receptor model, lidocaine will bind to both the resting state and the inactivated state and come to a new equilibrium (see inset to Fig. 5). Because affinity for the inactivated state is 50–100 times greater than for the resting state, and even though a very small fraction of channels are in the inactivated state initially, binding of lidocaine to the inactivated channels will draw channels to the bound inactivated state. To the extent that this happens, channels in the drug-bound inactivated state will be mis-interpreted as resting state block. This becomes crucially important in the investigations of tissue differences on the effects of differences in resting affinity because the voltage-dependent distribution between the resting state and the inactivated state (h{infty}) is much more negative in heart than it is in skeletal muscle (Table 1), thus, at any given holding potential, more channels are in the inactivated state for heart and the measurement of resting affinity will be artifactually increased for heart when compared with skeletal muscle. This effect would be predicted to decrease as holding potentials are made more negative relative to the h{infty} relationship because more channels are moved into the resting state.

We previously [6] measured tonic lidocaine block of native Na channels in rat ventricular cells and in mouse neuroblastoma cells at a holding potential of –150 mV (63 mV negative to the V1/2 for heart and 72 mV negative to the V1/2 for neuroblastoma for a 500-ms conditioning potential) and found a 2.3-fold greater tonic affinity for heart (1.8 versus 4.2 mM). Given that the slopes of the h{infty} relationship are generally similar (between 4.5 and 6.5), the V1/2 of the h{infty} relationship provides a convenient reference point to assess how negative the holding potential was to the inactivation process in previous studies. Nuss et al. [4] measured tonic block in hH1 and rSkM1 channels co-expressed with the β1-subunit in oocytes at a holding potential of –100 mV (only 20 mV negative to the V1/2 for heart and 40 mV negative to the V1/2 for skeletal muscle) and reported a 2.9-fold greater tonic affinity for heart (400 versus 1170 µM, respectively). Wang et al. [5] studied lidocaine block of hH1 and rSkM1 {alpha}-subunit channels expressed in a mammalian cell line (HEK293, tsa 201) and concluded that the cardiac sodium channel had an intrinsically higher resting affinity for lidocaine. They based this conclusion on the greater tonic block in heart at 100 µM lidocaine for a holding potential of –120 mV, and made no estimate of dissociation constants. The effect of the holding potential on resting affinity estimates was not assessed in any of these studies.

More recently, Wright et al. [8] studied cocaine and lidocaine affinities for human heart channels and rat skeletal muscle {alpha}-subunits expressed in a mammalian cell line (HEK293t) without co-expression of the β1-subunit and concluded that they had very similar resting affinities (440 and 491 µM for lidocaine, respectively) at –180 mV (86 mV negative to the V1/2 for heart and 102 mV negative to the V1/2 for skeletal muscle). From data at two lidocaine concentrations (30 and 300 µM), they showed that tonic block reached a plateau level for the two isoforms at –180 mV using a protocol with a 10-s conditioning step with a 100-ms recovery interval at –140 mV before applying the test step.

Our results contrast with those of Wright et al. [8] and show tonic block plateaus at significantly different levels as the membrane is hyperpolarized, with the affinity for hH1a being 4.8 times that of rSkM1(Fig. 4 and Table 1Go); this result supports the previous studies that suggested a higher intrinsic resting affinity for the heart {alpha}-subunit isoform. Several factors may account for the differences in our results from those of Wright et al. [8]. Our studies were done in oocytes and not mammalian cells. Inactivation kinetics of the {alpha}-subunit expressed in oocytes are different in the two expression systems, especially for rSkM1. In oocytes, decay of rSkM1 (but not hH1a) is abnormally slow without co-expression of β1, as seen in Fig. 1, and typical of previous studies (see [12,13] for review), whereas decay of the INa for the rSkM1 {alpha}-subunit in mammalian cells appears normal. In our present study in oocytes, co-expression of the β1-subunit with rSkM1 and hH1a narrowed the tissue isoform difference in affinity, although it did not eliminate it (Table 1). The β1-subunit can be detected by reverse transcriptase PCR in HEK cells (Kyle and Tonkovich, unpublished observations), suggesting that studies in cells such as those by Wright et al. [8] could possibly represent a β1-modified {alpha}-subunit.

The mechanism for β1-subunit effects on affinity of lidocaine for the {alpha}-subunit of the Na channel is not clear. The β1-subunit effects are unlikely to be purely through effects on inactivation kinetics because, if it were, one would then expect the plateau in apparent affinity at negative potentials to be equal. Also, the opposite effects on hH1 and rSkM1 would not be expected. The β1-subunit, therefore, appears to have an effect on lidocaine rest affinity that is independent of its effect on inactivation kinetics. Previously, we showed that a β1-subunit lacking the cytoplasmic tail had the same effect on resting affinity for hH1a as the intact subunit [14]. This, and the relatively small effect on affinity, suggests an indirect or allosteric change in the {alpha}-subunit caused by β1 rather than a direct interaction at the drug-binding site, which is presumed to be at a site in the internal pore [2,16].

This analysis has emphasized inactivation kinetics effects on lidocaine block because it is a property most affected by different {alpha} isoforms, the β1-subunit, and for affecting lidocaine affinity. A role for binding to ‘pre-open’ and to open states prior to reaching peak current can also be interpreted as tonic block [17]. Although isoform differences in activation kinetics for different {alpha} isoforms and for the β1-subunit are smaller than for inactivation, such differences might also affect estimation of lidocaine affinities for the {alpha}-subunit [9]. In addition, our long conditioning steps used to inactivate sodium channels very likely induced the channels into ‘slow’ inactivated states, which might have their own effects on drug block [18].

In conclusion, our data show that, when expressed in oocytes, the human Na-channel {alpha}-subunit has an intrinsically higher resting lidocaine affinity than that of the rat skeletal muscle isoform, and co-expression with the β1-subunit has opposite effects on affinity and also narrows this difference between isoforms. The differences in affinity are less than an order of magnitude and are likely to be small relative to differing ambient tissue conditions, such as resting potential, duty cycle and action potential duration in heart, skeletal muscle and nerve tissue. If, however, as suggested in ref. [19], β1-subunit expression is altered in development or in disease states, then this may have clinical significance. For studies of the underlying mechanism, however, this difference may be of biophysical significance in studies of local anesthetic binding to the voltage-gated Na channel [20] and have implications for channel structure–function.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by grants from the HL56441 (JCM) and HL20592 (JWK) and by the University of Wisconsin Cardiovascular Research Center and the Oscar Rennebohm Foundation.


   Notes
 
1 Present address: Department of Pharmacology and Physiology, University of Chicago, Chicago, IL 60637, USA. Back

2 Present address: Department of Physiology, University of Tennessee, College of Medicine, Memphis, TN 38163, USA. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Strichartz G.R. The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine. J Gen Physiol (1973) 62:37–57.[Abstract/Free Full Text]
  2. Ragsdale D.S, McPhee J.C, Scheuer T, Catterall W.A. Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science (1994) 265:1724–1728.[Abstract/Free Full Text]
  3. Satin J, Kyle J.W, Rogart R.B, et al. Lidocaine block of heart and brain Na channel isoforms. J Gen Physiol (1994) 104:18a. Abstract.
  4. Nuss H.B, Tomaselli G.F, Marban E. Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels. J Gen Physiol (1995) 106:1193–1209.[Abstract/Free Full Text]
  5. Wang D.W, Nie L, George A.L Jr., Bennett P.B. Distinct local anesthetic affinities in Na+ channel subtypes. Biophys J (1996) 70:1700–1708.[Abstract/Free Full Text]
  6. Makielski J.C. The heart sodium channel phenotype for inactivation and lidocaine block. Jpn Heart J (1996) 37:733–739.[Medline]
  7. Bean B.P, Cohen C.J, Tsien R.W. Lidocaine block of cardiac sodium channels. J Gen Physiol (1983) 81:613–642.[Abstract/Free Full Text]
  8. Wright S.N, Wang S.Y, Kallen R.G, Wang G.K. Differences in steady-state inactivation between Na channel isoforms affect local anesthetic binding affinity. Biophys J (1997) 73:779–788.[Abstract/Free Full Text]
  9. Nuss B.H, Kambouris N.G, Tomaselli G.F, et al. Sodium channel mutations that enhance fast modal gating induce lidocaine resistance. Circulation (1997) 96:I–120. Abstract.
  10. Hille B. Local anesthetics: hydrophilic and hydrophobic pathways for the drug–receptor reaction. J Gen Physiol (1977) 69:497–515.[Abstract/Free Full Text]
  11. Hondeghem L.M, Katzung B.G. Time- and age-dependent interactions of antiarrhythmic drugs with cardiac sodium channels. Biochim Biophys Acta (1977) 472:373–398. Review with 98 refs.[Medline]
  12. Isom L.L, De Jongh K.S, Catterall W.A. Auxiliary subunits of age-gated ion channels. Neuron (1994) 12:1183–1194. Review with 125 refs.[CrossRef][ISI][Medline]
  13. Goldin A.L. Accessory subunits and sodium channel inactivation. Curr Opin Neurobiol (1993) 3:272–277. Review with 54 refs.[CrossRef][Medline]
  14. Makielski J.C, Limberis J.T, Chang S.Y, Fan Z, Kyle J.W. Coexpression of beta 1 with cardiac sodium channel alpha-subunits in oocytes decreases lidocaine block. Mol Pharmacol (1996) 49:30–39.[Abstract]
  15. Makielski J.C, Limberis J, Fan Z, et al. Isoform and 1 subunit effects on lidocaine affinity for human heart and skeletal muscle Na channels expressed in oocytes. PACE (1997) 20:1217. Abstract.
  16. Hille B. Ionic channels of excitable membranes. (1992) second ed. Sunderland, MA: Sinauer Associates.
  17. Starmer C.F, Nesterenko V.V, Undrovinas A.I, Grant A.O, Rosenshtraukh L.V. Lidocaine blockade of continuously and transiently accessible sites in cardiac sodium channels. J Mol Cell Cardiol (1991) 23(Suppl_1):73–83.[CrossRef][ISI][Medline]
  18. Balser J.R, Nuss H.B, Romashko D.N, Marban E, Tomaselli G.F. Functional consequences of lidocaine binding to slow-inactivated sodium channels. J Gen Physiol (1996) 107:643–658.[Abstract/Free Full Text]
  19. Kupershmidt S, Yang T, Roden D.M. Modulation of cardiac Na+ current phenotype by β1-subunit expression. Circ Res (1998) 83:441–447.[Abstract/Free Full Text]
  20. Makielski J.C, Limberis J, Chen L, Boyce S. Inactivation and lidocaine block phenotypes in wild type and chimeric (heart and skeletal muscle) Na channels. Biophys J (1996) 70:A132. Abstract.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Makielski, J. C
Right arrow Articles by Kyle, J. W
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Makielski, J. C
Right arrow Articles by Kyle, J. W
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?