Accessibility of Site 1304 During Lidocaine Block
The major findings of this study are that (a
) lidocaine does not compete with the fast-inactivation gate, (b
) lidocaine potentiates the degree to which depolarization favors closure of the fast-inactivation gate, and (c
) lidocaine does not measurably affect the rate of recovery of the fast-inactivation gate. These observations were made possible by our ability to follow the position of the fast-inactivation gate with a conformational marker, the reactivity of site 1304 with MTS-ET, characterized in detail in a previous study (Vedantham and Cannon, 1998
In the first set of experiments, we determined the position of the fast-inactivation gate as a function of lidocaine concentration during tonic block, the inhibition of peak sodium current that occurs with infrequent depolarization. Our results indicate that at −120 mV, for lidocaine concentrations below the Kd for block, the majority of blocked channels are not fast- inactivated. Above the Kd for block, the data suggest that lidocaine favors closure of the fast-inactivation gate, although the certainty of this conclusion is undermined by the possibility of nonspecific effects interfering with the reaction between MTS-ET and site 1304 at such high drug concentrations. (Our data on the voltage dependence of the reaction rate show that nonspecific reduction of the reaction rate is not occurring at 1.0 mM lidocaine: the reaction rates in 1.0 mM lidocaine and control conditions are equal at very hyperpolarized voltages.)
Assuming that the modification rate faithfully reports the position of the fast-inactivation gate even above the Kd for tonic block, our observations on concentration dependence are consistent with state-dependent binding of lidocaine to channel conformations that are populated significantly only at depolarized potentials in the absence of drug. As the lidocaine concentration is increased, the population of channels that are in these “depolarized” conformations will increase by mass action, even at −120 mV. Because depolarized states favor closure of the fast-inactivation gate, increasing lidocaine concentration should also favor closure of the fast gate.
That the modification rate is reduced by 40–50% in the presence of 4.0 mM lidocaine at −120 mV predicts a dramatically altered h∞ curve: at −120 mV, a significant fraction of channels must be unavailable. We found, consistent with our data, that in 4.0 mM lidocaine, availability at −120 mV is somewhere on the steep portion of the h∞ curve, although we could not accurately estimate the relative availability at −120 mV because patches do not survive the strong hyperpolarizations (less than −140 mV) that would be required to determine the maximum availability (data not shown).
Our next set of experiments on the voltage dependence of site 1304 accessibility in the presence of lidocaine showed a 10.2-mV hyperpolarizing shift of the half-maximal modification rate, similar to the 10.8-mV hyperpolarizing shift of the V1/2 of the h∞ curve. However, Rmax and Rmin were not significantly changed, even though the maximum value of the h∞ curve was reduced by 22% in 1.0 mM lidocaine.
Most state-dependent models predict that block at very hyperpolarized voltages reflects binding of drug to noninactivated channels. Rmax, the limiting modification rate at such hyperpolarized voltages, reflects the position of the fully accessible fast-inactivation gate and should not, according to a state-dependent model, be reduced in the presence of 1.0 mM lidocaine, even if 22% of the channels are blocked. As the channels are depolarized, however, a state-dependent mechanism favors binding to channels further along in the activation pathway and predicts that the fraction of blocked channels that are fast-inactivated will increase. This explains the observed left shift of the voltage dependence of the modification rate. Rmin, which reflects the maximal degree of gate closure in F1304C, is not significantly changed in the presence of lidocaine, a finding that is also predicted by a state-dependent mechanism favoring inactivation.
The final set of experiments was directed at the effect of lidocaine on the recovery of site 1304 accessibility after brief depolarizing pulses. We first confirmed that 1.0 mM lidocaine dramatically slows the recovery of F1304C availability at −120 mV after a 20-ms depolarization to 0 mV. In the absence of lidocaine, the time constant of recovery is on the order of 1–2 ms, while in 1.0 mM lidocaine, it is ~100–200 ms. This effect produces use-dependent block, a frequency-dependent, cumulative inhibition of sodium current with repetitive depolarizations. Between 7.5 and 37.5 ms, only ~20– 30% of channels recover in the presence of lidocaine, whereas >90% recover with no lidocaine present. By contrast, the modification rate was not changed at all in the presence of 1.0 mM lidocaine, demonstrating that lidocaine does not significantly alter the kinetics of recovery from fast inactivation.
A Possible Mechanism of Lidocaine Action
At first glance, the results of these experiments seem to be in conflict: on the one hand, lidocaine shifts the h∞ curve in a way that favors fast inactivation, suggesting a stabilizing interaction between lidocaine block and fast-inactivated channels. On the other hand, lidocaine has no measurable effect on the off rate of the fast-inactivation particle, suggesting that it does not preferentially stabilize fast inactivation.
One model that reconciles our results is shown in Fig. . Following Kuo and Bean (1994)
, we employ a model for sodium channel gating consisting of several closed, noninactivated states, each in equilibrium with a fast-inactivated state (Fig. A). For convenience, only a few such equilibria are depicted. Horizontal equilibria represent the voltage-dependent transitions along the activation pathway, with depolarization favoring a rightward shift in the distribution of populated states. The vertical transitions, by contrast, are voltage independent, and the rightmost equilibria favor inactivated, rather than noninactivated, channels. According to this model, depolarization moves the distribution of channels to the right and down, while hyperpolarization tends to shift the distribution to the left and up.
Figure 8 A model for lidocaine action. In A, a section of the activation pathway for sodium channels is shown, in which each noninactivated state (Cn) is connected to an inactivated state (In). The length of the vertical arrows between inactivated and noninactivated (more ...)
Fig. B presents a qualitative model for how lidocaine affects the states depicted in Fig. A. We assume that each state can bind lidocaine, since our data suggest that both inactivated and noninactivated channels may experience block. We incorporate state dependence by postulating that lidocaine binds more favorably to channels that are further along in the activation pathway (towards the right), regardless of whether they are noninactivated or inactivated. In other words, lidocaine is sensitive to position along the horizontal, voltage-dependent axis of the state diagram, but not the vertical, voltage-independent axis. In this model, lidocaine does not directly affect the equilibrium constants between inactivated and noninactivated channels (the equilibrium distributions for Cn ↔ In and CnL ↔ InL are equal). Consequently, lidocaine binding does not affect the rate of recovery from fast inactivation by very much, in agreement with our findings on the recovery of accessibility of site 1304. However, the voltage-dependent equilibria in the activation pathway are altered in lidocaine-bound channels, shifting the overall distribution of channels to the right in Fig. B.
The model also explains why lidocaine causes a hyperpolarizing shift in the h∞ curve. By mass action, addition of lidocaine at any given voltage will tend to shift the distribution of channels towards the right in the state diagram of Fig. B. Since the vertical equilibria will favor fast-inactivated states as the distribution of channels moves sufficiently rightward along the activation pathway, the addition of lidocaine will indirectly promote fast inactivation. This phenomenon also explains our tonic block measurements: the greater the lidocaine concentration, the greater the rightward shift along the activation pathway, and hence the greater the fraction of inactivated channels.
The model also predicts a reciprocal effect of fast inactivation on lidocaine action: the presence of the fast-inactivation gate promotes block, because (like lidocaine) the fast-inactivation particle binds more tightly to the rightmost states on the activation pathway. This would partly explain why channels with disrupted fast inactivation show a reduction in sensitivity to lidocaine effects (Cahalan, 1978
; Yeh, 1978
; Bennett et al., 1995
; Balser et al., 1996
). We need not attribute this reduction in sensitivity to an essential role played by inactivation in the mechanism of lidocaine action.
Use-dependent block, in our model, is a consequence of a slow off rate of drug from the drug-bound, non–fast-inactivated states. Recall that at depolarized potentials, our data show that both lidocaine and the fast-inactivation particle are bound (i.e., the back, lower row in Fig B is populated), and that on repolarization the fast-inactivation particle dissociates rapidly, populating the back, upper row of Fig. B. The transitions from the back, upper row to the front, upper row, along with full leftward movement along the activation pathway, is rate limiting and slow (100-fold slower than recovery from fast inactivation), and generates use- dependent block when further depolarization occurs before full recovery.
A remaining question concerns the kinetics of leftward movement along the activation pathway upon repolarization. Because inactivation is not intrinsically voltage dependent, but derives its voltage dependence from activation, some leftward movement along the activation pathway must precede recovery from inactivation. In other words, some inward charge movement must occur if recovery from inactivation is to occur. Unfortunately, whether and to what extent lidocaine impedes inward charge movement upon repolarization has not been examined carefully. Our results predict that some component of the gating charge must remain relatively free to move even in lidocaine-bound channels, and that inward movement of this fraction must be sufficient for complete recovery of the fast- inactivation gate. Further experiments will be required to elucidate the details of the coupling between inactivation and gating charge movement in the presence of lidocaine, and thereby to determine how far the distribution of channels must move to the left on repolarization for full recovery from inactivation to occur.
Relation to Previous Work on Lidocaine
Our model is a version of the modulated receptor hypothesis (Hille, 1977
; Hondeghem and Katzung, 1977
), in which the affinity of a single receptor site for lidocaine is altered by the conformational state of the channel. Our model differs from Hille's original presentation and from that of Bean et al. (1983)
by not treating the inactivated state as the high-affinity state. Instead, we propose that transitions along the activation pathway (outward movement of S4 segments and/ or opening of the activation gate) affect the affinity of lidocaine for its receptor, following the proposals of Wang et al. (1987)
, Strichartz and Wang (1986)
, and Yeh and Tanguy, 1985
. Several lines of evidence support our hypothesis.
First, numerous studies have shown a reduction in the potency of local anesthetics in fast-inactivation defective sodium channels (Cahalan, 1978
; Yeh, 1978
; Bennett et al., 1995
; Balser et al., 1996
). However, despite the loss of potency, local anesthetics do retain their ability to generate tonic and use-dependent block in these channels (Shepley et al., 1983
; Strichartz and Wang, 1986
; Wang et al., 1987
). As noted above, this is consistent with the predictions of our model: the inactivation gate potentiates the effects of local anesthetics, but is not necessary to generate those effects. There is also evidence that at least some local anesthetic molecules can be trapped by closure of the activation gate, suggesting a possible mechanism for use-dependent block that does not involve the fast-inactivation gate (Strichartz, 1973
; Yeh and Tanguy, 1985
Gating-current studies have revealed that lidocaine can produce a hyperpolarizing shift in the Q
/V curve (Hanck et al., 1994
; Josephson and Chi, 1994
) along with a reduction in the total amount of on-gating current. A possible interpretation of this finding is that some of the voltage sensors of drug-bound channels move outward at less depolarized potentials than normal. This would entail, at any given voltage, a drug- induced rightward shift in the distribution of channels along the activation pathway diagrammed in Fig. , as our model predicts.
Finally, site-directed mutagenesis has placed the receptor for lidocaine roughly in the middle of the S6 transmembrane segment (Ragsdale et al., 1994
). Extrapolation to Na+
channels of a recent substituted cysteine accessibility study in segment S6 of Shaker
channels (Liu et al., 1997
) suggests that the position of the activation gate is likely to be very close to the local anesthetic binding site. Thus, it would not be surprising if the primary action of lidocaine is to interact with activation gating, perhaps by stabilizing the channel in the open conformation.
We wish to emphasize that our results are not sufficient to determine uniquely our particular model of lidocaine action. Although our results do suggest a very limited role for fast inactivation in generating use- dependent block, it is still possible that the affinity of lidocaine for its receptor is increased by closure of the fast-inactivation gate in the intact channel (i.e., with a phenylalanine at site 1304). Another possibility is that the Na+
channel slow inactivation mechanism plays a role in lidocaine action. Our finding that recovery from fast inactivation precedes recovery of the ionic current in the presence of lidocaine parallels an earlier finding that recovery from fast inactivation precedes recovery from slow inactivation (Vedantham and Cannon, 1998
), and raises the possibility that the two slowly recovering states are related in some way. For example, lidocaine might accelerate the rate of entry into slow-inactivated states.
Also, the mechanism of lidocaine action might vary among sodium channel isoforms. Our experiments were conducted in skeletal muscle sodium channels, which have a lower apparent lidocaine affinity that cardiac channels (Hille, 1978
; Nuss et al., 1995
). However, most of this difference is attributable to relative shifts in voltage-dependent gating between the two isoforms, rather than to differences in the putative binding site (Wright et al., 1997
), suggesting that our results with skeletal muscle channels will probably hold for cardiac channels as well. We should also emphasize that our results may not hold for all local anesthetics, which exhibit considerable variation at the chemical level as well as in their effects on sodium channels (Hille, 1977
These uncertainties aside, our data do enable us to place important new constraints on the possible forms that models for lidocaine action can take. Any such model must involve cooperativity between lidocaine binding and fast inactivation, and must incorporate a state that is slowly recovering, but not fast inactivated, to explain use-dependent block.