Localization-dependent activity of the Kv2.1 delayed-rectifier K+ channel

The Kv2.1 K⁺ channel is highly expressed throughout the brain, where it regulates excitability during periods of high-frequency stimulation. Kv2.1 is unique among Kv channels in that it targets to large surface clusters on the neuronal soma and proximal dendrites. These clusters also form in transfected HEK cells. Following excessive excitatory stimulation, Kv2.1 declusters with an accompanying 20- to 30-mV hyperpolarizing shift in the activation threshold. Although most Kv2.1 channels are clustered, there is a pool of Kv2.1 resident outside of these domains. Using the cell-attached patch clamp technique, we investigated the hypothesis that Kv2.1 activity varies as a function of cell surface location. We found that clustered Kv2.1 channels do not efficiently conduct K⁺, whereas the nonclustered channels are responsible for the high threshold delayed rectifier K⁺ current typical of Kv2.1. Comparison of gating and ionic currents indicates only 2% of the surface channels conduct, suggesting that the clustered channels still respond to membrane potential changes. Declustering induced via either actin depolymerization or alkaline phosphatase treatment did not increase whole-cell currents. Dephosphorylation resulted in a 25-mV hyperpolarizing shift, whereas actin depolymerization did not alter the activation midpoint. Taken together, these data demonstrate that clusters do not contain high threshold Kv2.1 channels whose voltage sensitivity shifts upon declustering; nor are they a reservoir of nonconducting channels that are activated upon release. On the basis of these findings, we propose unique roles for the clustered Kv2.1 that are independent of K⁺ conductance.     

mShal, a subfamily of A-type K+ channel cloned from mammalian brain.

We have cloned and expressed a mouse brain cDNA, mShal, that encodes a transient, A-type K+ current. mShal, the vertebrate homolog of the Drosophila Shal gene, defines a distinct subfamily of voltage-gated K+ channels. The Shal deduced proteins are more highly conserved between mouse and Drosophila than other presently known K+ channels. mShal carries a "low-threshold" A-type current with a hyperpolarized steady-state inactivation midpoint. Marked similarity was observed between mShal and its Drosophila homolog, fShal, with regard to voltage sensitivity of activation, macroscopic inactivation, steady-state inactivation, and 4-aminopyridine sensitivity. Sequence conservation for Shal proteins is unusually high at the amino terminus, an area considered important for inactivation. Removal of conserved amino-terminal residues from mShal modifies macroscopic inactivation but the transient nature of the current is preserved. Underlying the very high conservation of mShal and fShal may be a role in the nervous system that is conserved in widely divergent species.     

Single K+ channel currents of anomalous rectification in cultured rat myotubes.

The currents through single K+ channels of the anomalous (or inward) rectifier were recorded in tissue cultured rat myotubes by using the "gigohm seal" patch clamp technique developed by Sigworth and Neher. These unitary currents were detected as current fluctuations due to the blocking and unblocking of channels by Ba2+. The single-channel conductance was obtained from the slope of the linear relationship between unitary current amplitude and membrane potential. When the external solution contained 155 mM K+, the single-channel conductance was 10.4 +/- 2.6 pS (+/- SD; n = 6). This value was independent of the the concentration of blocking ions but increased with increasing external K+ concentration. The behavior of the unitary current agreed with that expected from the blocking kinetics of Ba2+ on the macroscopic K+ current of the anomalous rectifier. The density of the channel is likely to be small and may even be less than 1/micrometers 2.     

Early down-regulation of K+ channel genes and currents in the postinfarction heart

INTRODUCTION: Down-regulation of key K+ channel subunit gene expression and K+ currents is a universal response to cardiac hypertrophy, whatever the cause, including the postmyocardial infarction (post-MI) remodeled heart. METHODS AND RESULTS: We investigated the hypothesis that down-regulation of K+ channel genes and currents post-MI occurs early and before significant remodeled hypertrophy of the noninfarcted myocardium could be detected. We investigated (1) the incidence of induced ventricular tachyarrhythmias (VT) in 3-day post-MI rat heart; (2) action potential (AP) characteristics of isolated left ventricular (LV) myocytes from sham-operated and 3-day post-MI heart; (3) time course of changes in outward K+ currents Ito-fast(f) and I(K) in isolated myocytes from 3-day and 4-week post-MI noninfarcted LV and compared the changes with sham-operated animals; and (4) changes in the messenger and protein levels of Kv2.1, Kv4.2, and Kv4.3 in the LV and right ventricle of 3-day post-MI heart. Sustained VT was induced in 6 of 10 3-day post-MI rats and in none of 8 sham rats. The membrane capacitance of myocytes isolated from 3-day post-MI noninfarcted LV was not significantly different from control, whereas membrane capacitance 4-week post-MI was significantly higher, reflecting the development of hypertrophy. AP duration was increased and the density of Ito-f and I(K) were significantly decreased in 3-day post-MI LV myocytes compared with sham. The reduced density of Ito did not significantly differ in 4-week post-MI LV myocytes, whereas the density of I(K) was decreased further at 4 weeks post-MI. The changes in Ito-f and I(K) correlated with decreased messenger and protein levels of Kv4.2/Kv4.3 and Kv2.1, respectively. CONCLUSION: These results support the hypothesis that down-regulation of K+ channel gene expression and current in the post-MI LV occurs early and may be dissociated from the slower time course of post-MI remodeled hypertrophy. These changes may contribute to early arrhythmogenesis of the post-MI heart.     

External blockade of the major cardiac delayed-rectifier K+ channel (Kv1.5) by polyunsaturated fatty acids.

The present work shows that arachidonic acid and some other long chain polyunsaturated fatty acids such as docosahexaenoic acid, which is abundant in fish oil, produce a direct open channel block of the major voltage-dependent K+ channel (Kv1.5) cloned in cardiac cells. The inhibitory action of these selected fatty acids is seen when they are applied extracellularly but not when they are included in the patch pipette. Fatty acids then appear to bind to an external site on the Kv1.5 channel structure. Inhibition of Kv1.5 channel activity by polyunsaturated fatty acids (acceleration of the apparent inactivation and decrease of the peak current) is similar to that produced by the class III antiarrhythmic tedisamil. Docosahexaenoic acid and arachidonic acid also inhibit the delayed-rectifier K+ channel currents in cultured mouse and rat cardiomyocytes. These results are discussed in the light of the reported fatty acids effects on cardiac function in diseased states. Since Kv1.5 is also present in the brain, the results reported here could also have a significance in terms of processes such as long-term potentiation or depression.     

Extracellular K+ specifically modulates a rat brain K+ channel.

Extracellular potassium concentration is actively maintained within narrow limits in all higher organisms. Slight variations in extracellular potassium levels can induce major alterations of essential physiological functions in excitable tissues. Here we describe that superfusion of cultured rat hippocampal neurones with potassium-free medium leads to a decrease of a specific outward potassium current, probably carried by RCK4-type channels (RCK4 are potassium channels found in rat brain). This is confirmed by heterologous expression of these channels in Xenopus oocytes. In this system, variations of extracellular potassium in the physiological concentration range induce significant differences in current amplitude. Moreover, the current is completely suppressed in the absence of extracellular potassium. The potassium dependence of macroscopic conductance in RCK4 channels was related by site-directed mutagenesis to that lysine residue in the extracellular loop between the transmembrane segments S5 and S6 of RCK4 protein that confers resistance to extracellular blockage by tetraethylammonium. It is shown that extracellular potassium affects the number of available RCK4 channels, but not the single-channel conductance, the mean open time, or the gating charge displacement upon depolarization.     

Regulation of ROMK1 K+ channel activity involves phosphorylation processes.

An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.     

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.     

Gating charge interactions with the S1 segment during activation of a Na+ channel voltage sensor

Voltage-gated Na(+) channels initiate action potentials during electrical signaling in excitable cells. Opening and closing of the pore of voltage-gated ion channels are mechanically linked to voltage-driven outward movement of the positively charged S4 transmembrane segment in their voltage sensors. Disulfide locking of cysteine residues substituted for the outermost T0 and R1 gating-charge positions and a conserved negative charge (E43) at the extracellular end of the S1 segment of the bacterial Na(+) channel NaChBac detects molecular interactions that stabilize the resting state of the voltage sensor and define its conformation. Upon depolarization, the more inward gating charges R2 and R3 engage in these molecular interactions as the S4 segment moves outward to its intermediate and activated states. The R4 gating charge does not disulfide-lock with E43, suggesting an outer limit to its transmembrane movement. These molecular interactions reveal how the S4 gating charges are stabilized in the resting state and how their outward movement is catalyzed by interaction with negatively charged residues to effect pore opening and initiate electrical signaling.     

Benzocaine enhances and inhibits the K+ current through a human cardiac cloned channel (Kv1.5).

OBJECTIVE: The aim of this study was to analyze the effects of a neutral local anaesthetic, benzocaine, on a cardiac K+ channel cloned from human ventricle. METHODS: Experiments were performed on hKv1.5 channels stably expressed on mouse cells using the whole-cell configuration of the patch clamp technique. RESULTS: At 10 nM, benzocaine increased the current amplitude ("agonist effect") by shifting the activation curve 8.4 +/- 2.7 mV in the negative direction, and slowed the time course of tail current decline. In contrast, benzocaine (100-700 microM) inhibited hKv1.5 currents (KD = 901 +/- 81 microM), modified the voltage-dependence of channel activation, which became biphasic, and accelerated the channel deactivation. Extracellular K+ concentration ([K+]o) also affected the channel gating. At 140 mM [K+]o, the time course of tail currents deactivation was significantly accelerated, whereas at 0 mM [K+]o, it was slowed. At both [K+]o the activation curve became biphasic. Benzocaine accelerated the tail current decay at 0 mM but not at 140 mM [K+]o. The reduction in the permeation of K+ through the pore did not modify the blocking effects of micromolar concentrations of benzocaine, but suppressed the agonist effect observed at nanomolar concentrations. CONCLUSIONS: All these results suggest that benzocaine blocks and modifies the voltage- and time-dependent properties of hKv1.5 channels, binding to an extracellular and to an intracellular site at the channel level. Moreover, both sites are related to each other and can also interact with K+.     

Single mutations convert an outward K+ channel into an inward K+ channel

Shaker-type K(+) channels in plants display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, an Arabidopsis K(+) channel (SKOR) and a tomato K(+) channel (LKT1) share high amino acid sequence similarity and identical domain structures; however, SKOR conducts outward K(+) current and is activated by positive membrane potentials (depolarization), whereas LKT1 conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of SKOR and LKT1 remains unknown. Using a screening procedure combined with random mutagenesis, we identified in the SKOR channel single amino acid mutations that converted an outward-conducting channel into an inward-conducting channel. Further domain-swapping and random mutagenesis produced similar results, suggesting functional interactions between several regions of SKOR protein that lead to specific voltage-sensing properties. Dramatic changes in rectifying properties can be caused by single amino acid mutations, providing evidence that the inward and outward channels in the Shaker family from plants may derive from the same ancestor.     

K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.     

A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.     

Gating of the late Na+ channel in normal and failing human myocardium

We previously reported an ultraslow inactivating late Na+ current (INaL) in left ventricular cardiomyocytes (VC) isolated from normal (NVC) and failing (FVC) human hearts. This current could play a role in heart failure-induced repolarization abnormalities. To identify properties of NaCh contributing to INaL, we examined early and late openings in cell-attached patches of HEK293 cells expressing human cardiac NaCh alpha-subunit (alpha-HEK) and in VC of one normal and three failing human hearts. Two types of the late NaCh openings underlay INaL in all three preparations: scattered late (SLO) and bursts (BO). Amplitude analysis revealed that slope conductance for both SLO and BO was the same compared to the main level of early openings (EO) in both VC (21 vs 22.7pS, NVC; 22.7 vs 22.6pS, FVC) and alpha-HEK (23.2 vs 23pS), respectively. Analysis of SLO latencies revealed voltage-independent ultraslow inactivation in all preparations with tendency to be slower in FVC compared to NCV. EO and SLO render one open voltage-independent state (tau approximately 0.4ms) for NVC and FVC. One open (voltage-dependent) and two closed states (one voltage-dependent and another voltage-independent) were found in BO of both specimens. Burst duration tend to be longer in FVC ( approximately 50ms) than in NVC ( approximately 30ms). In FVC we found both modes SLO and BO at membrane potential of -10mV that is attribute for take-off voltages (from -18 to -2mV) for early afterdepolarizations (EAD's) in FVC. In conclusions, we found a novel gating mode SLO that manifest slow (hundreds of ms), voltage-independent inactivation in both NVC and FVC. We were unable to reliably demonstrate any differences in the properties of the late NaCh in failing vs a normal human heart. Accordingly, the late current appears to be generated by a single population of channels in normal and failing human ventricular myocardium. Both SLO and BO could be implicated in EADs in HF.     

Developmental changes in the functional characteristics and expression of voltage-gated K+ channel currents in rat aortic myocytes

OBJECTIVE: Active control of the arterial diameter by vascular smooth muscle is one of the principle mechanisms by which vessels adapt to a significant rise in blood pressure after birth. Although voltage-gated K+ (Kv) channels play an important role in the regulation of excitation-contraction coupling in arteries, very little is known about postnatal modification of Kv channels. We therefore investigated changes in the functional characteristics and expression of Kv channels in rat aortic myocytes (RAMs) during early postnatal development. METHODS: Kv currents (I(Kv)) were investigated in single smooth muscle cells freshly dispersed from neonatal (1-3 days) and adult Wistar rat thoracic aorta using the whole-cell patch clamp technique. RESULTS: I(Kv) in neonates had significantly faster activation kinetics and was inactivated at more positive voltages than I(Kv) in adults (half-inactivation potential -24+/-2 and -40+/-3 mV and slope factor 4.2+/-0.4 and 11.1+/-0.5 mV, respectively). No difference in the steady state activation was found. I(Kv) in neonates was insensitive to a high concentration of tetraethylammonium (TEA, 10 mM) but blocked 4-aminopyridine (4-AP, IC(50)=0.5+/-0.1 mM), whereas I(Kv) in adult RAMs was almost completely abolished by 10 mM TEA and was relatively insensitive to low concentrations of 4-AP. I(Kv) in both age groups was insensitive to charybdotoxin (300 nM) or alpha-dendrotoxin (200 nM). Immunoblot analysis showed that the expression of Kv1.2 alpha-protein decreased and Kv2.1 increased with development. CONCLUSION: Significant changes in functional characteristics of the native I(Kv) and the expression of particular Kv channel proteins occurred during postnatal vascular development. These changes could play an important role in adaptation to extrauterine life.     

Excitation-secretion coupling: ionic currents in glomerulosa cells: effects of adrenocorticotropin and K+ channel blockers

The ionic conductance of cultured rat glomerulosa cells has been studied using the whole cell variant of the patch-clamp technique. We have identified and partially characterized three currents: a transient outward current, a slow outward current, and a slow inward current. The transient outward current activated rapidly and then inactivated slowly on maintained depolarization. Activation was initiated at -30 mV, and zero current was seen at -60 to -50 mV. The slow outward current did not inactivate with time and was initiated around 0 mV; its zero current voltage was difficult to evaluate. The two outward currents were present in different proportions, which explains the different time course of the total outward current from one cell to another. A slow inward current was also found which activated near -30 mV and reached its reversal potential between 80 and 100 mV. This current was blocked by Co2+, increased with [Ca2+]o, and was insensitive to Na+-free external medium. ACTH, a potent stimulant of steroid output, was found to block the transient outward current, but was ineffective on the slow outward current and the slow inward current. Tetraethylammonium and 4-aminopyridine, K+ channel inhibitors, also blocked the transient outward current.     

An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis

The steroid hormone aldosterone is secreted both in the setting of intravascular volume depletion and hyperkalemia, raising the question of how the kidney maximizes NaCl reabsorption in the former state while maximizing K(+) secretion in the latter. Mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring increased renal NaCl reabsorption and impaired K(+) secretion. PHAII-mutant WNK4 achieves these effects by increasing activity of the Na-Cl cotransporter (NCC) and the Na(+) channel ENaC while concurrently inhibiting the renal outer medullary K(+) channel (ROMK). We now describe a functional state for WNK4 that promotes increased, rather than decreased, K(+) secretion. We show that WNK4 is phosphorylated by SGK1, a mediator of aldosterone signaling. Whereas wild-type WNK4 inhibits the activity of both ENaC and ROMK, a WNK4 mutation that mimics phosphorylation at the SGK1 site (WNK4(S1169D)) alleviates inhibition of both channels. The net result of these effects in the kidney would be increased K(+) secretion, because of both increased electrogenic Na(+) reabsorption and increased apical membrane K(+) permeability. Thus, modification at the PHAII and SGK1 sites in WNK4 impart opposite effects on K(+) secretion, decreasing or increasing ROMK activity and net K(+) secretion, respectively. This functional state for WNK4 would thus promote the desired physiologic response to hyperkalemia, and the fact that it is induced downstream of aldosterone signaling implicates WNK4 in the physiologic response to aldosterone with hyperkalemia. Together, the different states of WNK4 allow the kidney to provide distinct and appropriate integrated responses to intravascular volume depletion and hyperkalemia.     

The two-pore channel TPK1 gene encodes the vacuolar K+ conductance and plays a role in K+ homeostasis

The Arabidopsis thaliana genome contains five genes that encode two pore K+ (TPK) channels. The most abundantly expressed isoform of this family, TPK1, is expressed at the tonoplast where it mediates K+ -selective currents between cytoplasmic and vacuolar compartments. TPK1 open probability depends on both cytoplasmic Ca2+ and cytoplasmic pH but not on the tonoplast membrane voltage. The channel shows intrinsic rectification and can be blocked by Ba2+, tetraethylammonium, and quinine. TPK1 current was found in all shoot cell types and shows all of the hallmarks of the previously described vacuolar K (VK) tonoplast channel characterized in guard cells. Characterization of TPK1 loss-of-function mutants and TPK1-overexpressing plants shows that TPK1 has a role in intracellular K+ homeostasis affecting seedling growth at high and low ambient K+ levels. In stomata, TPK1 function is consistent with vacuolar K+ release, and removal of this channel leads to slower stomatal closure kinetics. During germination, TPK1 contributes to the radicle development through vacuolar K+ deposition to provide expansion growth or in the redistribution of essential minerals.     

A stretch-activated K+ channel sensitive to cell volume.

The role of K+ channels in cell osmoregulation was investigated by using the patch-clamp technique. In cell-attached patches from Necturus proximal tubule, the short-open-time K+ channel at the basolateral membrane could be stretch-activated by pipette suction, where a negative pressure of 6 cm H2O (588.6 Pa) was sufficient to increase the open probability of the channel by a factor of 4.0 +/- 0.8 (n = 7 tubules). A 50% reduction in bath osmolarity increased cell volume by 66 +/- 10% and increased the K+-channel open probability by a factor of 5.8 +/- 1.4 (n = 7) in the same cell-attached patches that were activated by pipette suction. A kinetic analysis indicates one open state and at least two closed states for this epithelial K+ channel. Both suction and swelling shorten the longest time constant of the closed-time distribution by a factor of 3, without significant effect on either the mean open time or the shorter closed-state time constant. The similar effect of suction and swelling is consistent with the hypothesis that stretch-activated K+ channels mediate the increase in macroscopic K+ conductance that occurs during osmoregulation of amphibian proximal tubules. Calculations based on a simple model indicate that small increments in cell volume could produce statistically significant increases in K+-channel activity.     

Electrostatic distance geometry in a K+ channel vestibule.

Many voltage-gated K+ channels carry in the external vestibule a receptor for charybdotoxin, a peptide channel blocker. We use point mutagenesis of both charybdotoxin and a Shaker K+ channel to isolate the electrostatic interaction energy between chosen pairs of residues, one on the channel and one on bound toxin. The results allow estimates of physical distances between such residue pairs and, in combination with the known structure of charybdotoxin, localize specific channel residues in three-dimensional space.