Binding of benzocaine in batrachotoxin-modified Na+ channels. State- dependent interactions

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX- modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use- dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.     

Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels.

Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.     

Altered stereoselectivity of cocaine and bupivacaine isomers in normal and batrachotoxin-modified Na+ channels

The inhibitory effects of local anesthetics (LAs) of cocaine and bupivacaine optical isomers on Na+ currents were studied in clonal GH3 cells under whole-cell patch clamp conditions. At holding potential of -100 mV, all four isomers inhibited peak Na+ currents when the cell was stimulated infrequently. The dose-response curves of this tonic block of peak Na+ currents by (-)/(+) cocaine and (-)/(+) bupivacaine were well fitted by the Langmuir isotherm, suggesting that one LA isomer blocked one Na+ channel. Each pair of isomers showed no greater than a twofold difference in stereoselectivity toward Na+ channels. Additional block of Na+ currents occurred when the cell was stimulated at 2 Hz. This use-dependent block was also observed in all four isomers, which again displayed little stereoselectivity. The voltage dependence of the use-dependent block produced by cocaine isomers did not overlap with the activation of Na+ channels but did overlap with the steady-state inactivation (h infinity), indicating that cocaine can bind directly to the inactivated state of Na+ channels before channel opening. In comparison, the peak batrachotoxin (BTX)-modified Na+ currents were little inhibited by cocaine and bupivacaine isomers. However, the maintained BTX-modified Na+ currents were highly sensitive toward the (-) form of cocaine and bupivacaine isomers during a prolonged depolarization. As a result, a profound time-dependent block of BTX-modified Na+ currents was evident in the presence of these LA isomers. The estimated values of the equilibrium dissociation constant (KD in micromolar) at +50 mV were 35.8, 661, 7.0, and 222 for (-)/(+) cocaine and (-)/(+) bupivacaine, respectively. Although chloramine-T (CT) also modified the fast inactivation of Na+ channels and gave rise to a maintained Na+ current during a prolonged depolarization, LA isomers showed no greater stereoselectivity in blocking this maintained current than in blocking the normal transient Na+ current. We conclude that (a) cocaine and bupivacaine isomers exhibit only weak stereoselectivity toward the LA receptor in normal and CT-treated Na+ channels, (b) BTX drastically modifies the configuration of the LA binding site so that the LA stereoselectivity of the open Na+ channels is altered by an order of magnitude, and (c) the (-) forms of cocaine and bupivacaine interact strongly with the open state of BTX-modified Na+ channels but only weakly, if at all, with the closed state. The last finding may explain why most LA drugs were reported to be less effective toward BTX-modified Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Inactivation of single cardiac Na+ channels in three different gating modes.

In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate.     

Modeling ion permeation through batrachotoxin-modified Na+ channels from rat skeletal muscle with a multi-ion pore.

The mechanism of ion permeation through Na+ channels that have been modified by batrachotoxin (BTX) and inserted into planar bilayers has been generally described by models based on single-ion occupancy, with or without an influence of negative surface charge, depending on the tissue source. For native Na+ channels there is evidence suggestive of a multi-ion conduction mechanism. To explore the question of ion occupancy, we have reexamined permeation of Na+, Li+, and K+ through BTX-modified Na+ channels from rat skeletal muscle. Single-channel current-voltage (I-V) behavior was studied in neutral lipid bilayers in the presence of symmetrical Na+ concentrations ranging from 0.5 to 3,000 mM. The dependence of unitary current on the mole fraction of Na+ was also examined in symmetrical mixtures of Na(+)-Li+ and Na(+)-K+ at a constant total ionic strength of 206 and 2,006 mM. The dependence of unitary conductance on symmetrical Na+ concentration does not exhibit Michaelis-Menten behavior characteristic of single-ion occupancy but can be simulated by an Eyring-type model with three barriers and two sites (3B2S) that includes double occupancy and ion-ion repulsion. Best-fit energy barrier profiles for Na+, Li+, and K+ were obtained by nonlinear curve fitting of I-V data using the 3B2S model. The Na(+)-Li+ and Na(+)-K+ mole-fraction experiments do not exhibit an anomalous mole-fraction effect. However, the 3B2S model is able to account for the biphasic dependence of unitary conductance on symmetrical [Na+] that is suggestive of multiple occupancy and the monotonic dependence of unitary current on the mole fraction of Na+ that is compatible with single or multiple occupancy. The best-fit 3B2S barrier profiles also successfully predict bi-ionic reversal potentials for Na(+)-Li+ and Na(+)-K+ in both orientations across the channel. Our experimental and modeling results reconcile the dual personality of ion permeation through Na+ channels, which can display features of single or multiple occupancy under various conditions. To a first approximation, the 3B2S model developed for this channel does not require corrections for vestibule surface charge. However, if negative surface charges of the protein do influence conduction, the conductance behavior in the limit of low [Na+] does not correspond to a Gouy-Chapman model of planar surface charge.     

Evidence for interactions between batrachotoxin-modified channels in hybrid neuroblastoma cells.

Current records from voltage-clamped membrane patches containing two batrachotoxin-modified sodium channels were analyzed to determine whether these channels are identical and independent. In most two-channel patches, the experimentally observed probabilities that zero, one, or two channels are open differ from the binomial distribution, demonstrating that the two channels are nonidentical or nonindependent or both. From the same current records, we also determined the rate for the transition from two open channels to one open channel and for the transition from one open channel to zero open channels. These data are consistent with closing rates for the two channels that are equal and independent. Both probability and closing rate data can be fit by a model wherein the channels are identical, the closing rates are independent, and the opening rate is greater when the other channel is closed than when it is open. The implications of this model for analyzing noise spectra and current variance are examined.     

Effect of a 125 mT static magnetic field on the kinetics of voltage activated Na+ channels in GH3 cells

Voltage activated Na(+) channels were examined in GH3 cells, using the whole cell patch clamp method. Channel currents were recorded before, during, and after a 150 s exposure to a 125 mT static magnetic field. There was a slight shift in the current-voltage relationship and a less than 5% reduction in peak current during magnetic field exposure. More pronounced, however, was an increase in the activation time constant, tau(m), during and for at least 100 s following exposure to the field. This change in tau(m) was seen primarily at lower activation voltages. No change was noted in the inactivation time constant, tau(h). Changes were clearly temperature dependent, being evident only at and above 35 degrees C. These findings are consistent with the hypothesis that reorientation of diamagnetic anisotropic molecules in the cell membrane are capable of distorting imbedded ion channels sufficiently to alter their function. The temperature dependence of this phenomenon is probably due to the greater ease with which a liquid crystal membrane can be deformed.     

Steady-state currents through voltage-dependent, dihydropyridine-sensitive Ca2+ channels in GH3 pituitary cells.

Ca2+ influx via voltage-dependent Ca2+ channels is known to be elicited during action potentials but possibly also occurs at the resting potential. The steady-state current through voltage-dependent Ca2+ channels and its role for the electrical activity was, therefore, investigated in pituitary GH3 cells. Applying the recently developed 'nystatin-modification' of the patch-clamp technique, most GH3 cells (18 out of 23 cells) fired spontaneous action potentials from a baseline membrane potential of 43.7 +/- 4.6 mV (mean +/- s.d., n = 23). The frequency of action potentials was stimulated about twofold by Bay K 8644 (100 nM), a Ca(2+)-channel stimulator, and action potentials were completely suppressed by the Ca(2+)-channel blocker PN 200-110 (100 nM). Voltage clamping GH3 cells at fixed potentials for several minutes and with 1 mM Ba2+ as divalent charge carrier, we observed steady-state Ca(2+)-channel currents that were dihydropyridine-sensitive and displayed a U-shaped current-voltage relation. The results strongly suggest that the observed long lasting, dihydropyridine-sensitive Ca(2+)-channel currents provide a steady-state conductivity for Ca2+ at the resting potential and are essential for the generation of action potentials in GH3 pituitary cells.     

Characterization of Ca2+-stimulated secretion in permeable GH3 pituitary cells

In this report, the secretory response to Ca2+ in GH3 rat pituitary cells permeabilized by electric field discharge has been compared in both magnitude and Ca2+ sensitivity to prolactin (PRL) release from intact GH3 cells. The half-maximally effective [Ca2+] for stimulating PRL release in permeable cells was approximately 0.5 microM, and maximal stimulation was obtained at 3-10 microM Ca2+. The magnitude of Ca2+ stimulation in permeable cells was in the same range as that obtained from an equal number of intact cells stimulated by depolarizing K+. Moreover, the Ca2+ sensitivity of PRL release in intact GH3 cells (measured by Quin 2 fluorescence) closely resembled the Ca2+ sensitivity determined in permeable cells. Release of a sulfated proteoglycan whose release is stimulated by secretagogues in intact cells was stimulated by Ca2+ in permeable cells with the same Ca2+ sensitivity as for PRL release. Maximal Ca2+ stimulation of PRL release in permeable cells required the addition of MgATP. Other energy sources (ADP, GTP, and inorganic phosphate) also supported Ca2+-stimulated secretion but were less effective. The above results indicated that PRL release from permeable cells resembles the physiological process in intact cells. The permeable cell system should prove useful in investigating the mechanism mediating the effect of Ca2+ on secretion, although our studies with pharmacological agents have so far proved inconclusive. Among calmodulin antagonists tested, only trifluoroperazine inhibited Ca2+-stimulated secretion, whereas pimozide and calmidazolium did not.     

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Gating of Na channels. Inactivation modifiers discriminate among models

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.     

Multimodal action of single Na+ channels in myocardial mouse cells.

Unitary Na+ currents of myocardial mouse cells were studied at room temperature in 10 cell-attached patches, each containing one and only one channel. Small-pore patch pipettes (resistance 10-97 M omega when filled with 200% Tyrode's solution) with exceptionally thick walls were used. Observed were both rapidly inactivating (6 patches) and slowly inactivating (3 patches) Na+ currents. In one patch, a slow transition from rather fast to slow inactivation was detected over a time of 0.5 h. A short and a long component of the open-channel life time were recorded at the beginning, but only a short one at the end of the experiment. Concomitantly, the first latency was slowed. Amplitude histograms showed that the electrochemical driving force across the pore of the channel did not change during this time. In three patches, a fast and repetitive switching between different modes of Na+ channel action could be clearly identified by plotting the long-time course of the averaged current per trace. The ensemble-averaged current formed in each mode was different in kinetics and amplitude. Each mode had a characteristic mean open-channel life time and distribution of first latency, but the predominant single-channel current amplitude was unaffected by mode switches. It is concluded that two types of changes in kinetics may happen in a single Na+ channel: fast and reversible switches between different modes, and a slow loss of inactivation.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Polyamines block Ca2+-activated K+ channels in pituitary tumor cells (GH3)

The effects of the natural polyamines, putrescine, spermidine and spermine on single calcium-activated potassium channels from clonal rat pituitary tumor cells (GH3) were studied. Applied to inside-out patches, polyamines were found to reduce the current amplitude and open probability of the channels in a dose- and voltage-dependent manner, indicating that polyamines act as fast blockers which sense a fraction of the electrical field in the channel pore. The K _d for spermine was 11.2 mm for the reduction of unitary current amplitude and 0.7 mm for the reduction of the open probability. The order of effectiveness was spermine > spermidine > putrescine. From fitting -functions to current amplitude histograms, blocking and unblocking rates were determined as 11.4 × 10⁴ sec^–1 and 21.9 × 10⁴ sec^–1, respectively. The reduction of the channel open probability was relieved by an increase of the Ca²⁺ concentration of the internal solution, indicating that polyamines compete with Ca²⁺ at the Ca²⁺ sensor of the channel. Putrescine antagonized the effect of spermine on the channel current amplitude. The results suggest that polyamines at intracellular millimolar concentrations suppress ion channel activity and therefore may effect electrical discharge behavior of excitable cells.This work was supported in part by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung, P8587.