External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving approximately 37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located approximately 13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.     

Interaction between quaternary ammonium ions in the pore of potassium channels. Evidence against an electrostatic repulsion mechanism

We have examined the interaction between internal and external ions in the pore of potassium channels. We found that external tetraethylammonium was able to antagonize block of Shaker channels by internal TEA when the external and internal solutions contained K(+) ions. This antagonism was absent in solutions with Rb(+) as the only permeant ion. An externally applied trivalent TEA analogue, gallamine, was less effective than the monovalent TEA in inhibiting block by internal TEA. In addition, block by external TEA was little affected by changes in the concentration of internal K(+) ions, but was increased by the presence of internal Na(+) ions in the pore. These results demonstrate that external and internal TEA ions, likely located at opposite ends of the pore selectivity filter, do not experience a mutual electrostatic repulsion. We found that these results can be simulated by a simple 4-barrier-3-site permeation model in which ions compete for available binding sites without long-range electrostatic interactions.     

Repulsion between tetraethylammonium ions in cloned voltage-gated potassium channels.

Tetraethylammonium ion (TEA+) blocks voltage-gated K+ channels by acting at two sites located at opposite ends of the aqueous pore. This allowed us to test two predictions made by models of ion permeation, namely that K+ channels can be simultaneously occupied by multiple ions and that the ions repel each other. We show that externally applied TEA+ antagonize block by internal TEA+ and vice versa. The antagonism is less than predicted for competitive binding, hence TEA+ may occupy both sites simultaneously. External TEA+ and internal TEA+ reduce each others affinity 4- to 5-fold. In addition, K+ antagonizes block by TEA+ at the opposite side of the membrane, and external TEA+ antagonizes is block by internal Ba2+. The antagonism between ions applied at opposite sides of the membrane may be common to all cations binding to K+ channels.     

The multi-ion nature of the pore in Shaker K+ channels.

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.     

Two stable, conducting conformations of the selectivity filter in Shaker K+ channels

We have examined the voltage dependence of external TEA block of Shaker K(+) channels over a range of internal K(+) concentrations from 2 to 135 mM. We found that the concentration dependence of external TEA block in low internal K(+) solutions could not be described by a single TEA binding affinity. The deviation from a single TEA binding isotherm was increased at more depolarized membrane voltages. The data were well described by a two-component binding scheme representing two, relatively stable populations of conducting channels that differ in their affinity for external TEA. The relative proportion of these two populations was not much affected by membrane voltage but did depend on the internal K(+) concentration. Low internal K(+) promoted an increase in the fraction of channels with a low TEA affinity. The voltage dependence of the apparent high-affinity TEA binding constant depended on the internal K(+) concentration, becoming almost voltage independent in 5 mM. The K(+) sensitivity of these low- and high-affinity TEA states suggests that they may represent one- and two-ion occupancy states of the selectivity filter, consistent with recent crystallographic results from the bacterial KcsA K(+) channel. We therefore analyzed these data in terms of such a model and found a large (almost 14-fold) difference between the intrinsic TEA affinity of the one-ion and two-ion modes. According to this analysis, the single ion in the one-ion mode (at 0 mV) prefers the inner end of the selectivity filter twofold more than the outer end. This distribution does not change with internal K(+). The two ions in the two-ion mode prefer to occupy the inner end of the selectivity filter at low K(+), but high internal K(+) promotes increased occupancy of the outer sites. Our analysis further suggests that the four K(+) sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.     

Ion-Ion interactions at the selectivity filter. Evidence from K(+)-dependent modulation of tetraethylammonium efficacy in Kv2.1 potassium channels

In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations 相似文献   

Inhibition of the collapse of the Shaker K+ conductance by specific scorpion toxins

The Shaker B K(+) conductance (G(K)) collapses when the channels are closed (deactivated) in Na(+) solutions that lack K(+) ions. Also, it is known that external TEA (TEA(o)) impedes the collapse of G(K), and that channel block by TEA(o) and scorpion toxins are two mutually exclusive events. Therefore, we tested the ability of scorpion toxins to inhibit the collapse of G(K) in 0 K(+). We have found that these toxins are not uniform regarding the capacity to protect G(K). Those toxins, whose binding to the channels is destabilized by external K(+), are also effective inhibitors of the collapse of G(K). In addition to K(+), other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K(+) channels. The inhibition of the drop of G(K) follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K(+) current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect G(K) in 0 K(+) do so by interacting either with the most external K(+) binding site of the selectivity filter (suggesting that the K(+) occupancy of only that site of the pore may be enough to preserve G(K)) or with sites capable of binding K(+) located in the outer vestibule of the pore, above the selectivity filter.     

The block of Shaker K+ channels by kappa-conotoxin PVIIA is state dependent.

kappa-conotoxin PVIIA is the first conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents. We studied the mechanism of inhibition and concluded that PVIIA blocks the ion pore with a 1:1 stoichiometry and that binding to open or closed channels is very different. Open-channel properties are revealed by relaxations of partial block during step depolarizations, whereas double-pulse protocols characterize the slower reequilibration of closed-channel binding. In 2.5 mM-[K+]o, the IC50 rises from a tonic value of approximately 50 to approximately 200 nM during openings at 0 mV, and it increases e-fold for about every 40-mV increase in voltage. The change involves mainly the voltage dependence and a 20-fold increase at 0 mV of the rate of PVIIA dissociation, but also a fivefold increase of the association rate. PVIIA binding to Shaker Delta6-46 channels lacking N-type inactivation or to wild phenotypes appears similar, but inactivation partially protects the latter from open-channel unblock. Raising [K+]o to 115 mM has little effect on open-channel binding, but increases almost 10-fold the tonic IC50 of PVIIA due to a decrease by the same factor of the toxin rate of association to closed channels. In analogy with charybdotoxin block, we attribute the acceleration of PVIIA dissociation from open channels to the voltage-dependent occupancy by K+ ions of a site at the outer end of the conducting pore. We also argue that the occupancy of this site by external cations antagonizes on binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. It is concluded that PVIIA can also be a valuable tool for probing the state of ion permeation inside the pore.     

Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel.

To understand the role of permeating ions in determining blocking ion-induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.     

Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore.

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = approximately 265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30-100 microM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.     

Anomalous effects of external TEA on permeation and gating of the A-type potassium current in H. aspersa neuronal somata

The model proposed for external TEA block of Shaker K+ channels predicts a proportional relationship between TEA sensitivity and calculated electrical distance derived from measurements of voltage dependence of TEA block. In the present study, we examined this relationship for the A-type K+ current (IA) of Helix aspersa in neuronal somata using the whole-cell patch-clamp technique. External TEA inhibited IA with strong voltage dependence, such that the TEA dissociation constant was increased at depolarized test potentials. The half-inhibition constant (V0.5) for TEA block was approximately 21 mM at 0 mV, and V0.5 increased to approximately 67 mM at 50 mV. The calculated electrical distance for TEA block suggested that TEA traversed 65% of the way into the membrane electrical field. TEA also caused significant shifts in the voltage-dependence of A-type K+ channel gating. For example, at TEA concentrations below that required to fully suppress delayed outward currents, TEA caused depolarizing shifts in the voltage-dependence of A-type channel activation, steady-state inactivation, time for removal of inactivation, and slowed channel activation kinetics. Taken together, these observations suggest that TEA biased the local field potential near voltage-sensing domains of A-type K+ channels, causing the transmembrane electrical field to be relatively hyperpolarized in the presence of TEA. In summary, the calculated electrical distance of TEA block of A-type K+ channels in H. aspersa neurons is unprecedented among other K+ channels. This raises concerns about the conventional interpretation of this value. Furthermore, the voltage-dependent properties of IA are modified by TEA at concentrations previously used to isolate delayed rectifier potassium channels (IKDR) selectively. This lack of specificity has important implications for recent, as well as future studies of IA in H. aspersa and possibly other snail neurons.     

K(+) occupancy of the N-methyl-d-aspartate receptor channel probed by Mg(2+) block

The single-channel kinetics of extracellular Mg(2+) block was used to probe K(+) binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels. K(+) binds to three sites: two that are external and one that is internal to the site of Mg(2+) block. The internal site is approximately 0.84 through the electric field from the extracellular surface. The equilibrium dissociation constant for this site for K(+) is 304 mM at 0 mV and with Mg(2+) in the pore. The occupancy of any one of the three sites by K(+) effectively prevents the association of extracellular Mg(2+). Occupancy of the internal site also prevents Mg(2+) permeation and increases (by approximately sevenfold) the rate constant for Mg(2+) dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at -60 mV, there is approximately 1,400-fold apparent decrease in the affinity of the channel for extracellular Mg(2+) and approximately 2-fold enhancement of the apparent voltage dependence of Mg(2+) block caused by the voltage dependence of K(+) occupancy of the external and internal sites.     

Inactivation and pharmacological properties of sqKv1A homotetramers in Xenopus oocytes cannot account for behavior of the squid "delayed rectifier" K(+) conductance

Considerable published evidence suggests that alpha-subunits of the cloned channel sqKv1A compose the "delayed rectifier" in the squid giant axon system, but discrepancies regarding inactivation properties of cloned versus native channels exist. In this paper we define the mechanism of inactivation for sqKv1A channels in Xenopus oocytes to investigate these and other discrepancies. Inactivation of sqKv1A in Xenopus oocytes was found to be unaffected by genetic truncation of the N-terminus, but highly sensitive to certain amino acid substitutions around the external mouth of the pore. External TEA and K(+) ions slowed inactivation of sqKv1A channels in oocytes, and chloramine T (Chl-T) accelerated inactivation. These features are all consistent with a C-type inactivation mechanism as defined for Shaker B channels. Treatment of native channels in giant fiber lobe neurons with TEA or high K(+) does not slow inactivation, nor does Chl-T accelerate it. Pharmacological differences between the two channel types were also found for 4-aminopyridine (4AP). SqKv1A's affinity for 4AP was poor at rest and increased after activation, whereas 4AP block occurred much more readily at rest with native channels than when they were activated. These results suggest that important structural differences between sqKv1A homotetramers and native squid channels are likely to exist around the external and internal mouths of the pore.     

Modulation of potassium channel gating by external divalent cations

We have examined the actions of Zn2+ ions on Shaker K channels. We found that low (100 microM) concentrations of Zn2+ produced a substantial (approximately three-fold) slowing of the kinetics of macroscopic activation and inactivation. Channel deactivation was much less affected. These results were obtained in the presence of 5 mM Mg2+ and 4 mM Ca2+ in the external solution and so are unlikely to be due to modification of membrane surface charges. Furthermore, the action of 100 microM Zn2+ on activation was equivalent to a 70-mV reduction of a negative surface potential whereas the effects on deactivation would require a 15-mV increase in surface potential. External H+ ions reduced the Zn-induced slowing of macroscopic activation with an apparent pK of 7.3. Treatment of Shaker K channels with the amino group reagent, trinitrobenzene sulfonic acid (TNBS), substantially reduced the effects of Zn2+. All these results are qualitatively similar to the actions of Zn2+ on squid K channels, indicating that the binding site may be a common motif in potassium channels. Studies of single Shaker channel properties showed that Zn2+ ions had little or no effect on the open channel current level or on the open channel lifetime. Rather, Zn2+ substantially delayed the time to first channel opening. Thus, K channels appear to contain a site to which divalent cations bind and in so doing act to slow one or more of the rate constants controlling transitions among closed conformational states of the channel.     

Evidence for sequential ion-binding loci along the inner pore of the IRK1 inward-rectifier K+ channel

Steep rectification in IRK1 (Kir2.1) inward-rectifier K(+) channels reflects strong voltage dependence (valence of approximately 5) of channel block by intracellular cationic blockers such as the polyamine spermine. The observed voltage dependence primarily results from displacement, by spermine, of up to five K(+) ions across the narrow K(+) selectivity filter, along which the transmembrane voltage drops steeply. Spermine first binds, with modest voltage dependence, at a shallow site where it encounters the innermost K(+) ion and impedes conduction. From there, spermine can proceed to a deeper site, displacing several more K(+) ions and thereby producing most of the observed voltage dependence. Since in the deeper blocked state the leading amine group of spermine reaches into the cavity region (internal to the selectivity filter) and interacts with residue D172, its trailing end is expected to be near M183. Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter. As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A. For technical simplicity, we used tetraethylammonium (TEA) as an initial probe to test whether the corresponding residue in IRK1, F254, forms the shallower site. We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine. These results establish the evidence for physically separate, sequential ion-binding loci along the long inner pore of IRK1, and strongly suggest that the aromatic side chains of F254 underlie the likely innermost binding locus for both blocker and K(+) ions in the cytoplasmic pore.     

Shortening of the period of the circadian rhythm by a K+ channel blocker, tetraethylammonium, in the duckweed Lemna gibba G3

The period of the circadian rhythm of uptake of K+ by Lemna gibba strain G3 (duckweed), cultured in a flow medium, was shortened by continuous application of 0.5 mM tetraethylammonium chloride (TEA), which functions as a K+ channel blocker in both animal and plant cells. Other quaternary ammonium ions shortened the period of the rhythm in direct proportion to their ability to block K+ channels in cells of the nervous system. Ca2+ appears to be specific in its ability to antagonize this action of TEA and of its analogues.     

Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels.     

External tetraethylammonium as a molecular caliper for sensing the shape of the outer vestibule of potassium channels

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

A marine snail neurotoxin shares with scorpion toxins a convergent mechanism of blockade on the pore of voltage-gated K channels.

kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in kappa-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, kappa-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) M-1 s-1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced kappa-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the alpha-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.