Block of L-type calcium channels by charged dihydropyridines. Sensitivity to side of application and calcium.

We have studied block of L-type calcium channels by intracellular and extracellular application of the ionized dihydropyridine derivatives amlodipine and SDZ 207-180. We find that extracellular application of either drug causes voltage-dependent block of calcium channels. However, neither drug is effective when applied intracellularly. The insensitivity of calcium channels to intracellular drug is not due to the low concentrations of cytosolic calcium, because voltage-dependent block by ionized amlodipine, SDZ 207-180, and the neutral drug nisoldipine persists under conditions in which Ca0 is buffered by EGTA. In fact, the time course of the development of block by the ionized but not neutral drug molecules studied, is slower in the presence than in the absence of calcium. Our results indicate that the DHP binding site of the L-type calcium channel is close to the extracellular surface of the cell membrane and that ionized DHP molecules may interact with the receptor in a manner that is uniquely affected by calcium.     

Alterations in dihydropyridine receptors in dystrophin-deficient cardiac muscle

The deficiency of dystrophin, a critical membrane stabilizing protein, in the mdx mouse causes an elevation in intracellular calcium in myocytes. One mechanism that could elicit increases in intracellular calcium is enhanced influx via the L-type calcium channels. This study investigated the effects of the dihydropyridines BAY K 8644 and nifedipine and alterations in dihydropyridine receptors in dystrophin-deficient mdx hearts. A lower force of contraction and a reduced potency of extracellular calcium (P < 0.05) were evident in mdx left atria. The dihydropyridine agonist BAY K 8644 and antagonist nifedipine had 2.7- and 1.9-fold lower potencies in contracting left atria (P < 0.05). This corresponded with a 2.0-fold reduction in dihydropyridine receptor affinity evident from radioligand binding studies of mdx ventricular homogenates (P < 0.05). Increased ventricular dihydropyridine receptor protein was evident from both radioligand binding studies and Western blot analysis and was accompanied by increased mRNA levels (P < 0.05). Patch-clamp studies in isolated ventricular myocytes showed no change in L-type calcium current density but revealed delayed channel inactivation (P < 0.05). This study indicates that a deficiency of dystrophin leads to changes in dihydropyridine receptors and L-type calcium channel properties that may contribute to enhanced calcium influx. Increased influx is a potential mechanism for the calcium overload observed in dystrophin-deficient cardiac muscle.     

Mechanism of K+ channel block by verapamil and related compounds in rat alveolar epithelial cells

The mechanism by which the phenylalkylamines, verapamil and D600, and related compounds, block inactivating delayed rectifier K+ currents in rat alveolar epithelial cells, was investigated using whole-cell tight- seal recording. Block by phenylalkylamines added to the bath resembles state-dependent block of squid K+ channels by internally applied quarternary ammonium ions (Armstrong, C.M. 1971. Journal of General Physiology. 58:413-437): open channels are blocked preferentially, increased [K+]o accelerates recovery from block, and recovery occurs mainly through the open state. Slow recovery from block is attributed to the existence of a blocked-inactivated state, because recovery was faster in three situations where recovery from inactivation is faster: (a) at high [K+]o, (b) at more negative potentials, and (c) in cells with type l K+ channels, which recover rapidly from inactivation. The block rate was used as a bioassay to reveal the effective concentration of drug at the block site. When external pH, pHo, was varied, block was much faster at pHo 10 than pHo 7.4, and very slow at pHo 4.5. The block rate was directly proportional to the concentration of neutral drug in the bath, suggesting that externally applied drug must enter the membrane in neutral form to reach the block site. High internal pH (pHi 10) reduced the apparent potency of externally applied phenylalkylamines, suggesting that the cationic form of these drugs blocks K+ channels at an internal site. The permanently charged analogue D890 blocked more potently when added to the pipette than to the bath. However, lowering pHi to 5.5 did not enhance block by external drug, and tertiary phenylalkylamines added to the pipette solution blocked weakly. This result can be explained if drug diffuses out of the cell faster than it is delivered from the pipette, the block site is reached preferentially via hydrophobic pathways, or both. Together, the data indicate the neutral membrane-bound drug blocks K+ channels more potently than intracellular cationic drug. Neutral drug has rapid access to the receptor, where block is stabilized by protonation of the drug from the internal solution. In summary, externally applied phenylalkylamines block open or inactivated K+ channels by partitioning into the cell membrane in neutral form and are stabilized at the block site by protonation.     

Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle.

Sodium currents were studied under voltage clamp in the presence of neutral, amine, and quaternary local anesthetic compounds. Use-dependent block was observed as a cumulative depression of INa seen with repetitive depolarizing test pulses applied at frequencies of 2-10s-1. With quaternary QX-314, the time constant of use dependence was long, and with neutral benzocaine, very short. With lidocaine and procaine, increasing external pH (pHo) changed the time constant from long to short, but alterations of internal pH have no effect. Inactivation in Na channels was measured by the influence of prepulses on peak INa during test pulses. Single-stimulus inactivation curves were shifted more with lidocaine at high pHo than at low pHo, but inactivation curves measured during pulse trains with any of the drugs and at any pHo were strongly shifted. All measurements show that the drug-receptor reaction was slow for amine drugs at low pHo, as for quaternary drugs at any pHo, and fast for amine drugs at high pHo, as for neutral drugs at any pHo. The major effect of low pHo on amine drugs was to reduce the concentration of drugs in the fiber and to protonate drug molecules on the receptor, thus trapping them in the blocking position for a longer time. Direct effects of pH on the receptor seemed minimal.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, approximately 50% neutral at pH 7.4) have similar structures but markedly different effects on Na(+) channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na(+) channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na(+) channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na(+) channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na(+) channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.     

Local anesthetics: hydrophilic and hydrophobic pathways for the drug- receptor reaction

The properties of Na channels of the node of Ranvier are altered by neutral, amine, and quaternary local anesthetic compounds. The kinetics of the Na currents are governed by a composite of voltage- and time-dependent gating processes with voltage- and time-dependent block of channels by drug. Conventional measurements of steady-state sodium inactivation by use of 50-ms prepulses show a large negative voltage shift of the inactivation curve with neutral benzocaine and with some ionizable amines like lidocaine and tetracaine, but no shift is seen with quaternary OX-572. However, when the experiment is done with repetitive application of a prepulse-testpulse waveform, a shift with the quaternary cations (applied internally) is seen as well. 1-min hyperpolarizations of lidocaine- or tetracaine-treated fibers restore two to four times as many channels to the conducting pool as 50-ms hyperpolarizations. Raising the external Ca++ concentration also has a strong unblocking effect. These manipulations do not relieve block in fibers treated with internal quaternary drugs. The results are interpreted in terms of a single receptor in Na channels for the different drug types. Lipid-soluble drug forms are thought to come and go from the receptor via a hydrophobic region of the membrane, while charged and less lipid-soluble forms pass via a hydrophilic region (the inner channel mouth). The hydrophilic pathway is open only when the gates of the channel are open. Any drug form in the channel increases the probability of closing the inactivation gate which, in effect, is equivalent to a negative shift of the voltage dependence of inactivation.     

Calcicludine binding to the outer pore of L-type calcium channels is allosterically coupled to dihydropyridine binding

How dihydropyridines modulate L-type voltage-gated Ca2+ channels is not known. Dihydropyridines bind cooperatively with Ca2+ binding to the selectivity filter, suggesting that they alter channel activity by promoting structural rearrangements in the pore. We used radioligand binding and patch-clamp electrophysiology to demonstrate that calcicludine, a toxin from the venom of the green mamba snake, binds in the outer vestibule of the pore and, like Ca2+, is a positive modulator of dihydropyridine binding. Data were fit using an allosteric scheme where dissociation constants for dihydropyridine and calcicludine binding, KDHP and KCaC, are linked via the coupling factor, alpha. Nine acidic amino acids located within the S5-Pore-helix segment of repeat III were sequentially changed to alanine in groups of three, resulting in the mutant channels, Mut-A, Mut-B, and Mut-C. Mut-A, whose substitutions are proximal to IIIS5, exhibits a 4.5-fold reduction in dihydropyridine binding and is insensitive to calcicludine binding. Block of Mut-A currents by calcicludine is indistinguishable from wild-type, indicating that KCaC is unchanged and that the coupling between dihydropyridine and calcicludine binding (i.e., alpha) is disrupted. Mut-B and Mut-C possess KDHP values that resemble that of the wild type. Mut-C, the most C-terminal of the mutant channels, is insensitive to calcicludine binding and block. KCaC values for the Mut-C single mutants, E1122A, D1127A, and D1129A, increase from 0.3 (wild type) to 1.14, 2.00, and 20.5 microM, respectively. Together, these findings suggest that dihydropyridine antagonist and calcicludine binding to L-type Ca2+ channels promote similar structural changes in the pore that stabilize the channel in a nonconducting, blocked state.     

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.     

Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.

Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.     

Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells

Inhibition of whole-cell calcium currents in enzymatically dispersed frog atrial myocytes by D-600, diltiazem, and nifedipine was studied using a single-micropipette voltage-clamp technique. The objective of these experiments was to test the applicability of a modulated-receptor hypothesis similar to that proposed for local anesthetic interactions with sodium channels to account for the tonic and frequency-dependent interactions of these organic compounds with myocardial calcium channels. Data consistent with such a hypothesis include: (a) prominent use-dependent block of iCa by D-600 and diltiazem, which are predominantly charged at physiological pH; (b) iCa block by an externally applied, permanently charged dihydropyridine derivative is greatly attenuated; (c) all three antagonists produce large negative shifts in the voltage dependence of iCa availability; (d) block of iCa by these compounds is state-dependent; (e) reactivation of iCa in the presence of all three antagonists is biexponential, which suggests that drug-free channels recover with a normal time course and drug-bound channels recover more slowly; and (f) the kinetics of the drug-induced slow iCa recovery process may be determined largely by factors such as size and molecular weight, in addition to lipid solubility of the compounds. Experiments in which the pH was modified, however, reveal some important differences for the interaction of organic calcium antagonists with myocardial calcium channels. Acidification, in addition to changing the proportion of charged and neutral antagonist in solution, was found to selectively antagonize tonic inhibition of iCa by diltiazem and nifedipine, without changing the kinetics of the drug-induced slow iCa reactivation process. It is concluded that two distinct receptor sites may be involved in block of iCa by some of these compounds: a proton-accessible site and a proton-inaccessible site.     

Dissecting lidocaine action: diethylamide and phenol mimic separate modes of lidocaine block of sodium channels from heart and skeletal muscle.

We have investigated block of sodium channels by diethylamide and phenol, which resemble the hydrophilic tertiary amine head and the hydrophobic aromatic tail of the lidocaine molecule, respectively. Diethylamide and phenol separately mimicked the fast and slow modes of block caused by lidocaine. Experiments were performed using single batrachotoxin-activated bovine cardiac and rat skeletal muscle sodium channels incorporated into neutral planar lipid bilayers. Diethylamide, only from the intracellular side, caused a voltage-dependent reduction in apparent single channel amplitude ('fast' block). Block was similar for cardiac and skeletal muscle channels, and increased in potency when extracellular sodium was replaced by N-methylglucamine, consistent with an intrapore blocking site. Thus, although occurring at 15-fold higher concentrations, block by diethylamide closely resembles the fast mode of block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80-90). For cardiac sodium channels, phenol bound to a closed state causing the appearance of long blocked events whose duration increased with phenol concentration. This slow block depended neither on voltage nor on the side of application, and disappeared upon treatment of the channel with trypsin. For skeletal muscle channels, slow phenol block occurred with only very low probability. Thus, phenol block resembles the slow mode of block observed for lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:91-100). Our data suggest that there are separate sites for fast lidocaine block of the open channel and slow block of the "inactivated" channel. Fast block by diethylamide inhibited the long, spontaneous, trypsin-sensitive (inactivation-like) closures of cardiac channels, and hence secondarily antagonized slow block by phenol or lidocaine. This antagonism would potentiate shifts in the balance between the two modes of action of a tertiary amine drug caused by changes in the relative concentrations of the charged (fast blocking) and neutral (slow blocking) forms of the drug.     

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.     

Separation of dihydropyridine binding sites from cardiac junctional sarcoplasmic reticulum

When microsomes from feline ventricular muscle are centrifuged on continuous linear sucrose gradients, the major peak for the distribution pattern of the dihydropyridine binding sites corresponds in position and shape with the distribution of the Mr 300K polypeptide marker for junctional sarcoplasmic reticulum (SR). Plasma membrane vesicles are also present in those gradient fractions and appear to be joined to the junctional SR as native dyads. We now report that when such putative dyads are passed through the French press, both the dihydropyridine binding sites and the plasma membrane marker band together at a new isopycnic point distinct from the junctional SR. We conclude that as has been found in the skeletal muscle system the dihydropyridine binding sites are a marker for the junctional domain of the plasma membrane and that separation of the dyad components of the mammalian myocardium can be attained.     

Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes

Skeletal and cardiac dihydropyridine receptors function both as voltage- dependent L-type calcium channels (L-channels) and as critical proteins that trigger calcium release from the sarcoplasmic reticulum in muscle. In spite of these similarities, skeletal L-channels exhibit a markedly slower activation rate than cardiac L-channels. We investigated the mechanisms underlying this difference by comparing the unitary behavior of L-channels in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, or chimeric dihydropyridine receptors. Our results demonstrate that ensemble averages activate rapidly for the purely cardiac dihydropyridine receptor and approximately five times more slowly for L-channels attributable to the purely skeletal dihydropyridine receptor or a chimeric dihydropyridine receptor in which only the first internal repeat and all of the putative intracellular loops are of skeletal origin. All of the constructs studied similarly exhibit a brief (2-ms) and a long (> or = 15-ms) open time in the presence of Bay K 8644, neither of which depend significantly on voltage. In the absence of Bay K 8644, the fraction of total open events is markedly shifted to the briefer open time without altering the rate of ensemble activation. Closed time analysis of L- channels with cardiac-like, rapid activation (recorded in the presence of dihydropyridine agonist) reveals both a brief (approximately 1-ms) closed time and a second, voltage-dependent, long-lasting closed time. The time until first opening after depolarization is three to six times faster for rapidly activating L-channels than for slowly activating L- channels and depends strongly on voltage for both types of channels. The results suggest that a voltage-dependent, closed-closed transition that is fast in cardiac L-channels and slow in skeletal L-channels can account for the difference in activation rate between these two channels.     

Interaction of barium ions with potassium channels in squid giant axons.

Blocking of potassium channels by internally and externally applied barium ions has been studied in squid giant axons. Internal Ba (3-5 mM) causes rapid decay or "inactivation" of potassium current (IK). The kinetics and degree of block are strongly voltage-dependent. Large positive voltages speed blocking and make it more profound. Raising the external potassium concentration (Ko) from 0 to 250 mM has the opposite effect: block is made slower and less severe. In contrast, for positive voltages block by the tetraethylammonium derivative 3-phenylpropyltriethylammonium ion is almost independent of Ko and voltage. Recovery from block by internal Ba has a rapid phase lasting a few milliseconds and a slow phase lasting approximately 5 min. Internal Ba causes a "hook" in the IK tails recorded on repolarizing the fiber in high potassium external medium. External Ba, on the other hand, blocks without much altering IK time-course. KD (the dissociation constant) for block by external Ba is a few millimolar, and depends on the internal potassium concentration, the holding potential, and other factors. A reaction scheme for Ba and K channels is presented, postulating that internal and external Ba reach the same point in the channel. Once there, Ba blocks and also stabilizes the closed conformation of the channel. The extreme stability of the Ba channel complex implies the existence of negative charge within the channel.     

Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C). The interaction is restricted to the CB and IQ motifs involved in the calmodulin-mediated Ca(2+)-dependent inactivation of the DHPR, suggesting functional interactions between the two channels.     

The pH-dependent rate of action of local anesthetics on the node of Ranvier

Local anesthetic solutions were applied suddenly to the outside of single myelinated nerve fibers to measure the time course of development of block of sodium channels. Sodium currents were measured under voltage clamp with test pulses applied several times per second during the solution change. The rate of block was studied by using drugs of different lipid solubility and of different charge type, and the external pH was varied from pH 8.3 to pH 6 to change the degree of ionization of the amine compounds. At pH 8.3 the half-time of action of amine anesthetics such as lidocaine, procaine, tetracaine, and others was always less than 2 s and usually less than 1 s. Lowering the pH to 6.0 decreased the apparent potency and slowed the rate of action of these drugs. The rate of action of neutral benzocaine was fast (1 s) and pH independent. The rate of action of cationic quaternary QX-572 was slow (greater than 200 s) and also pH independent. Other quaternary anesthetic derivatives showed no action when applied outside. The result is that neutral drug forms act much more rapidly than charged ones, suggesting that externally applied local anesthetics must cross a hydrophobic barrier to reach their receptor. A model representing diffusion of drug into the nerve fiber gives reasonable time courses of action and reasonable membrane permeability coefficients on the assumption that the hydrophobic barrier is the nodal membrane. Arguments are given that there may be a need for reinterpretation of many published experiments on the location of the anesthetic receptor and on which charge form of the drug is active to take into account the effects of unstirred layers, high membrane permeability, and high lipid solubility.