Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer's disease therapy.

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

Differential actions of insecticides on target sites: basis for selective toxicity.

Whereas the selective toxicity of insecticides between insects and mammals has a long history of studies, it is now becoming abundantly clear that, in many cases, the differential action of insecticides on insects and mammalian target receptor sites is an important factor. In this paper, we first introduce the mechanism of action and the selective toxicity of pyrethroids as a prototype of study. Then, a more detailed account is given for fipronil, based primarily on our recent studies. Pyrethroids keep the sodium channels open for a prolonged period of time, causing elevation of the depolarizing after-potential. Once the after-potential reaches the threshold for excitation, repetitive after-discharges are produced, resulting in hyperexcitation of intoxicated animals. Only about 1% of sodium channels needs to be modified to produce hyperexcitation, indicating a high degree of toxicity amplification from sodium channels to animals. Pyrethroids were >1000-fold more potent on cockroach sodium channels than rat sodium channels, and this forms the most significant factor to explain the selective toxicity of pyrethroids in insects over mammals. Fipronil, a phenylpyrazole, is known to act on the gamma-aminobutyric acid receptor to block the chloride channel. It is effective against certain species of insects that have become resistant to most insecticides, including those acting on the gamma-aminobutyric acid receptor, and is much more toxic to insects than to mammals. Recently, fipronil has been found to block glutamate-activated chloride channels in cockroach neurons in a potent manner. Since mammals are devoid of this type of chloride channel, fipronil block of the glutamate-activated chloride channel is deemed responsible, at least partially, for the higher selective toxicity to insects over mammals and for the lack of cross-resistance.

Dynamics of 9-aminoacridine block of sodium channels in squid axons.

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency-dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9-aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.

Kinetics of 9-aminoacridine block of single Na channels.

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

Interactions of monovalent cations with sodium channels in squid axon. I. Modification of physiological inactivation gating.

Inactivation of Na channels has been studied in voltage-clamped, internally perfused squid giant axons during changes in the ionic composition of the intracellular solution. Peak Na currents are reduced when tetramethylammonium ions (TMA+) are substituted for Cs ions internally. The reduction reflects a rapid, voltage-dependent block of a site in the channel by TMA+. The estimated fractional electrical distance for the site is 10% of the channel length from the internal surface. Na tail currents are slowed by TMA+ and exhibit kinetics similar to those seen during certain drug treatments. Steady state INa is simultaneously increased by TMA+, resulting in a "cross-over" of current traces with those in Cs+ and in greatly diminished inactivation at positive membrane potentials. Despite the effect on steady state inactivation, the time constants for entry into and exit from the inactivated state are not significantly different in TMA+ and Cs+. Increasing intracellular Na also reduces steady state inactivation in a dose-dependent manner. Ratios of steady state INa to peak INa vary from approximately 0.14 in Cs+- or K+-perfused axons to approximately 0.4 in TMA+- or Na+-perfused axons. These results are consistent with a scheme in which TMA+ or Na+ can interact with a binding site near the inner channel surface that may also be a binding or coordinating site for a natural inactivation particle. A simple competition between the ions and an inactivation particle is, however, not sufficient to account for the increase in steady state INa, and changes in the inactivation process itself must accompany the interaction of TMA+ and Na+ with the channel.

Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating.

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.

BTX modification of Na channels in squid axons. I. State dependence of BTX action.

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.

Kinetic analysis of pancuronium interaction with sodium channels in squid axon membranes.

The interaction of pancuronium with sodium channels was investigated in squid axons. Sodium current turns on normally but turns off more quickly than the control with pancuronium 0.1-1mM present internally; The sodium tail current associated with repolarization exhibits an initial hook and then decays more slowly than the control. Pancuronium induces inactivation after the sodium inactivation has been removed by internal perfusion of pronase. Such pancuronium-induced sodium inactivation follows a single exponential time course, suggesting first order kinetics which represents the interaction of the pancuronium molecule with the open sodium channel. The rate constant of association k with the binding site is independent of the membrane potential ranging from 0 to 80 mV, but increases with increasing internal concentration of pancuronium. However, the rate constant of dissociation l is independent of internal concentration of pancuronium but decreases with increasing the membrane potential. The voltage dependence of l is not affected by changine external sodium concentration, suggesting a current-independent conductance block, The steady-state block depends on the membrane potential, being more pronounced with increasing depolarization, and is accounted for in terms of the voltage dependence of l. A kinetic model, based on the experimental observations and the assumption on binding kinetics of pancuronium with the open sodium channel, successfully simulates many features of sodium current in the presence of pancuronium.

Dynamics of aminopyridine block of potassium channels in squid axon membrane.

Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequency-dependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley n-type gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.

Interaction of n-alkylguanidines with the sodium channels of squid axon membrane.

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.

Endplate channel block by guanidine derivatives.

The effects of the n-alkyl derivatives of guanidine on the frog neuromuscular junction were studied using the two-microelectrode voltage clamp and other electrophysiological techniques. Methyl-, ethyl-, and propylguanidine stimulated the nerve-evoked release of transmitter. However, amyl-and octylguanidine had no apparent presynaptic action. All of the derivatives blocked the postsynaptic response to acetylcholine, the potency sequence being octyl-greater than amyl-greater than propyl-, methyl-greater than ethylguanidine. Methyl- and octylguanidine did not protect the receptor from alpha-bungarotoxin block, suggesting that these compounds do not bind to the receptor but probably block the ionic channel. Methyl-, ethyl-, and propylguanidine shortened inward endplate currents but prolonged outward currents. Amylguanidine prolonged both inward and outward endplate currents, and the currents became biphasic at negative membrane potentials. Octylguanidine increased the rate of decay of endplate currents at all potentials. All of the derivatives blocked inward endplate currents more markedly than outward currents, resulting in a highly nonlinear current-voltage relation. Methyl-, ethyl-, and propylguanidine reversed the voltage dependence of endplate current decay, while amyl-and octylguanidine reduced the voltage dependence of endplate current decay. Octylguanidine appears to block the ionic channel in both the open and the closed state. The block of the open channel follows pseudo-first-order kinetics with a forward rate constant of 4-6 X 10(7) M-1 s-1.

Current-dependent block of endplate channels by guanidine derivatives.

Methyl- and ethylguanidine block the endplate current in frog muscle. Both derivatives blocked inward-going endplate currents without affecting outward endplate currents. Repetitive stimulation that evoked several inward endplate currents enhanced the block, which suggests that these agents interact with open endplate channels. The relative conductance vs. potential curve exhibited a transition from a low to a high value near the reversal potential for the endplate current, both in normal and in 50% Na solution. In the latter solution, the reversal potential for endplate current was shifted by a mean value of 16 mV in the direction of hyperpolarization. The results suggest that methyl- and ethylguanidine block open endplate channels in a manner dependent on the direction of current flow rather than on the membrane potential.

Post-stroke dementia. Nootropic drug modulation of neuronal nicotinic acetylcholine receptors.

Nefiracetam is a new pyrrolidone nootropic drug that is being developed for clinical use in the treatment of post-stroke vascular-type and Alzheimer's-type dementia. Among a few neuroreceptors that have been identified as potential targets of nootropics, neuronal nicotinic acetylcholine receptors (nnAChRs) are deemed the most important since they are related to learning, memory, and Alzheimer's disease dementia. We have recently found potent stimulating action of nefiracetam on nnAChRs. Rat cortical neurons in long-term primary culture expressed nnAChRs. Whole-cell patch clamp experiments revealed two types of currents induced by ACh, alpha-bungarotoxin (alpha-BuTX)-sensitive, rapidly desensitizing, alpha 7-type currents and alpha-BuTX-insensitive, slowly desensitizing, alpha 4 beta 2-type currents. Although alpha 7-type currents were only weakly inhibited by nefiracetam, alpha 4 beta 2-type currents were potently and efficaciously potentiated by nefiracetam. Nefiracetam at 0.1 nM reversibly potentiated ACh-induced currents to 200-300% of control. Very high concentrations (about 10 microM) also potentiated these currents, but to a lesser extent, indicative of the bell-shaped dose-response relationship known to occur for nefiracetam, even in animal behavior experiments. Three specific inhibitors of each of PKA and PKC did not prevent nefiracetam from potentiating ACh-induced currents, indicating that these protein kinases are not involved in nefiracetam action. Pretreatment with pertussis toxin did not alter nefiracetam potentiation, indicating Gi/Go proteins are not involved. Pretreatment with cholera toxin did abolish nefiracetam potentiation. Thus, nefiracetam potentiation is mediated via Gs proteins. In conclusion, nefiracetam stimulates alpha 4 beta 2-type nnAChRs via Gs proteins at nanomolar concentrations. The potentiation of alpha 4 beta 2-type nnAChRs is thought to be at least partially responsible for cognitive enhancing action.

Modulation of Outward K(+) Conductance Is a Post-Activational Event in Rat T Lymphocytes Responsible for the Adoptive Transfer of Experimental Allergic Encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an accepted animal model for the human demyelinating disease multiple sclerosis. The continuously propagated line of Lewis rat T helper lymphocytes (GP1 T cells), specific for the encephalitogenic 68-86 sequence of guinea pig myelin basic protein (GPMBP), mediates the adoptive transfer of EAE into normal syngeneic Lewis rats. Because mitogenic activation of T cells can increase K(+) conductance, this study investigated changes in the outwardly rectifying K(+) conductance in GP1 T cells following activation with the encephalitogen, GPMBP. Using the gigohm.seal whole-cell variation of the patch clamp technique, GP1 T cells were studied during a 3-day culture with GPMBP and throughout the subsequent 10 days, as cells progressed through both GPMBP-induced activation (EAE transfer activity) and proliferation responses, finally reverting to the resting state. Resting GP1 T cells exhibited peak K(+) conductances around 2 nS, while GPMBP-induced activation resulted in 5- to 10-fold increases in peak K(+) conductance, which temporally coincided with the optimal period for EAE transfer activity. During and immediately after the optimal period for EAE transfer, 20-mV depolarizing shifts in the voltage dependence of both activation and inactivation developed, abruptly reversing to resting values as cells reverted to the resting state. Accompanying the depolarizing shifts were a slowing of the K(+) current activation kinetics and an acceleration of the deactivation kinetics. These results indicate that the K(+) conductance in GP1 rat T helper cells is modulated over the full time course of GPMBP-induced cellular responses and that K(+) channels should be optimally available during the period of adoptive EAE transfer, preceding disease manifestation. Copyright 1997 S. Karger AG, Basel

Potassium Channel Blockers Inhibit Adoptive Transfer of Experimental Allergic Encephalomyelitis by Myelin-Basic-Protein-Stimulated Rat T Lymphocytes.

Agents which block T cell K(+) currents can prohibit both proliferative and effector cell functions in T cells activated by mitogens or phorbol esters. This study examined the effects of some of these blocking agents on the immune responsiveness of guinea pig myelin basic protein (GPMBP)-reactive Lewis rat T lymphocytes, which are capable of mediating the adoptive transfer of experimental allergic encephalomyelitis (EAE), an accepted animal model for multiple sclerosis. Both the proliferative functions (DNA synthesis and cell blastogenesis) and the EAE transfer activities of GPMBP-reactive lymphocytes were examined following GPMBP-induced activation in the presence of agents shown to block the outwardly rectifying K(+) current in these cells. At concentrations which completely inhibited DNA synthesis, as measured by [(3)H]thymidine incorporation, and cell blastogenesis, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and methoxyverapamil (D60) completely blocked the subsequent adoptive transfer of EAE into naive syngeneic Lewis rats. The concentrations at which these blockers produced a 50% reduction in DNA synthesis were estimated to be 16, 1.6 and 32 &mgr;M for TEA, 4-AP and D-600, respectively, which were roughly equivalent to the EC(50) to block the K(+) current. Apamine, a potent Ca(2+)-activated K(+) channel blocker, at a concentration several orders of magnitude higher than is necessary to block Ca(2+)-activated K(+) channels, reduced the maximal K(+) conductance in GPMBP-reactive T cell K(+) channels by about 20%, but did not alter either [H(3)H]thymidine incorporation or the adoptive transfer of EAE. These results indicate that delayed rectifier K(+) channel blockers may prevent the activation of GPMBP-reactive T cells, thus prohibiting encephalitogenic effector cell functions. Copyright 1997 S. Karger AG, Basel

Differential block of two types of sodium channels by anticonvulsants.

The differential effects of the anticonvulsants phenytoin and carbamazepine on the sodium channels of rat dorsal root ganglion (DRG) neurons were studied using the patch clamp technique. The action potentials from tetrodotoxin-resistant (TTX-R) cells were less sensitive to phenytoin and carbamazepine than those from tetrodotoxin-sensitive (TTX-S) cells. The steady-state inactivation curve for TTX-S sodium channels was shifted by as much as 20.6 mV and 11.4 mV by phenytoin and carbamazepine at 300 microM, respectively, yet the curve for TTX-R sodium channels was shifted only by 6.0 mV and 6.9 mV, respectively. Thus, the differential action potential block of TTX-S and TTX-R cells by phenytoin and carbamazepine is due to different voltage dependence of and differential drug effects on the inactivation kinetics of two types of sodium channels.

Ion permeation and selectivity of squid axon sodium channels modified by tetramethrin.

The pyrethroid tetramethrin greatly prolongs the sodium current during step depolarization and the sodium tail current associated with step repolarization of the squid axon membrane. Non-linear current-voltage relationships for the sodium tail current were analyzed to assess the open sodium channel properties which included the permeation of various cations, calcium block and cation selectivity. Tetramethrin had no effect on any of these properties. It was concluded that tetramethrin modifies the sodium channel gating machinery without affecting the pore properties.

G-proteins are involved in riluzole inhibition of high voltage-activated calcium channels in rat dorsal root ganglion neurons.

Effects of riluzole on high voltage-activated (HVA) calcium channels of rat dorsal root ganglion neurons were studied using the whole-cell patch-clamp technique. Riluzole at 30 microM inhibited the HVA currents. The onset and offset of riluzole inhibitory effect were slow usually taking more than 3 min. Riluzole inhibition of the HVA currents was abolished and partially reduced by addition of 500 microM GDP-beta-S and 1 mM N-ethylmaleimide, respectively, to the pipette solution. Pre-treatment with pertussis toxin or application of depolarizing pre-pulses did not affect riluzole's inhibitory effect on the HVA currents. Riluzole inhibition of the HVA currents was also blocked by internal application of 50 microg/ml protein kinase A inhibitory peptide. It was concluded that pertussis toxin-insensitive G-proteins and protein kinase A may be involved in riluzole inhibition of the HVA currents.

Inactivation gating and 4-AP sensitivity in human brain Kv1.4 potassium channel.

Voltage-gated K(+) channels vary in sensitivity to block by 4-aminopyridine (4-AP) over a 1000-fold range. Most K(+) channel phenotypes with leucine at the fourth position (L4) in the leucine heptad repeat region, spanning the S4-S5 linker, exhibit low 4-AP sensitivity, while channels with phenylalanine exhibit high sensitivity. Mutational analysis on delayed rectifier type K(+) channels demonstrate increased 4-AP sensitivity upon mutation of the L4 heptad leucine to phenylalanine. This mutation can also influence inactivation gating, which is known to compete with 4-AP in rapidly inactivating A-type K(+) channels. Here, in a rapidly inactivating human brain Kv1.4 channel, we demonstrate a 400-fold increase in 4-AP sensitivity following substitution of L4 with phenylalanine. Accompanying this mutation is a slowing of inactivation, an acceleration of deactivation, and depolarizing shifts in the voltage dependence of activation and steady-state inactivation. To test the relative role of fast inactivation in modulating 4-AP block, N-terminal deletions of the fast inactivation gate were carried out in both channels. These deletions produced no change in 4-AP sensitivity in the mutant channel and approximately a six-fold increase in the wild type channel. These results support the view that changes at L4 which increase 4-AP sensitivity are largely due to 4-AP binding and may, in part, arise from alterations in channel conformation. Primarily, this study demonstrates that the fast inactivation gate is not a critical determinant of 4-AP sensitivity in Kv1.4 channels.

All or none block of single Na+ channels by tetrodotoxin.

The effects of tetrodotoxin on single Na+-channel currents recorded from excised patches of neuroblastoma cells were examined. Tetrodotoxin was found to cause a dose-dependent reduction in the frequency at which Na+ channels conduct during a series of depolarizations. Surviving conducting states had normal open times and current amplitudes. These effects could be explained by a model which includes initial binding of tetrodotoxin to a closed state of the channel with stable, complete block during the time the channel would normally be gated open.