Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels

In this study, single-channel recordings of high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)-blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed-blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.     

Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of alpha-subunit tyrosine 190 affect both binding and gating.

Affinity labeling and mutagenesis studies have demonstrated that the conserved tyrosine Y190 of the acetylcholine receptor (AChR) alpha-subunit is a key determinant of the agonist binding site. Here we describe the binding and gating kinetics of embryonic mouse AChRs with mutations at Y190. In Y190F the dissociation constant for ACh binding to closed channels was reduced approximately 35-fold at the first binding site and only approximately 2-fold at the second site. At both binding sites the association and dissociation rate constants were decreased by the mutation. Compared with wildtype AChRs, doubly-liganded alpha Y190F receptors open 400 times more slowly but close only 2 times more rapidly. Considering the overall activation reaction (vacant-closed to fully occupied-open), there is an increase of approximately 6.4 kcal/mol caused by the Y-to-F mutation, of which at least 2.1 and 0.3 kcal/mol comes from altered agonist binding to the first and second binding sites, respectively. The closing rate constant of alpha Y190F receptors was the same with ACh, carbamoylcholine, or tetramethylammonium as the agonist. This rate constant was approximately 3 times faster in ACh-activated S, W, and T mutants. The equilibrium dissociation constant for channel block by ACh was approximately 2-fold lower in alpha Y190F receptors compared with in wildtype receptors, suggesting that there are changes in the pore region of the receptor as a consequence of the mutation. The activation reaction is discussed with regard to energy provided by agonist-receptor binding contacts, and by the intrinsic folding energy of the receptor.     

Probing an open CFTR pore with organic anion blockers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.     

Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K+ channel

A series of n-alkyl-bis-alpha,omega-trimethylammonium (bisQn) compounds was synthesized, and their ability to block K+ currents through a K+ channel from sarcoplasmic reticulum was studied. K+ channels were inserted into planar phospholipid membranes, and single-channel K+ currents were measured in the presence of the blocking cations. These bisQn compounds block K+ currents only from the side of the membrane opposite to the addition of SR vesicles (the trans side). The block is dependent on transmembrane voltage, and the effective valence of the block (a measure of this voltage dependence) varies with the methylene chain length. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 1.1 to 0.65; it then remains constant at approximately 0.65 for bisQ5 to bisQ8; the effective valence abruptly increases to 1.2-1.3 for chains of nine carbons and longer. For the compounds of nine carbons and longer, the discrete nature of the block can be observed directly as 'flickering noise" on the open channel. The kinetics of the block were studied for these long-chain blockers. Both blocking and unblocking rates of the blockers vary with chain length, with the blocking rate showing the strongest variation--an increase of 2.8-fold per added methylene group. All of the voltage dependence of the binding equilibrium resides in the blocking rate, and none in the unblocking rate. The results imply that 65% of the voltage drop within the channel occurs over a distance of 6-7A, and that the short-chain blockers bind in a bent-over conformation with both charges deeply inside the channel.     

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.     

Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.     

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment.

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.     

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.     

Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle

The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include Na channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and Na channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 degrees C. The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property is a conserved feature of channel function for batrachotoxin-activated channels. The decrease in binding affinity for TTX and STX in low-affinity subtypes is due to a 3-9-fold decrease in the association rate constant and a 4-8-fold increase in the dissociation rate constant. For a series of STX derivatives, the association rate constant for toxin binding is approximately an exponential function of net toxin charge in membranes of neutral lipids, implying that there is a negative surface potential due to fixed negative charges in the vicinity of the toxin receptor. The magnitude of this surface potential (-35 to -43 mV at 0.2 M NaCl) is similar for both high- and low-affinity subtypes, suggesting that the lower association rate of toxin binding to toxin-insensitive subtypes is not due to decreased surface charge but rather to a slower protein conformational step. The increased rates of toxin dissociation from insensitive subtypes can be attributed to the loss of a few specific bonding interactions in the binding site such as loss of a hydrogen bond with the N-1 hydroxyl group of neosaxitoxin, which contributes about 1 kcal/mol of intrinsic binding energy.     

Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers

Lipid metabolites, free fatty acids and lysophospholipids, modify the function of membrane proteins including ion channels. Such alterations can occur through signal transduction pathways, but may also result from "direct" effects of the metabolite on the protein. To investigate possible mechanisms for such direct effects, we examined the alterations of gramicidin channel function by lysophospholipids (LPLs): lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). The experiments were done on planar bilayers formed by diphytanoylphosphatidylcholine in n-decane a system where receptor- mediated effects can be excluded. At aqueous concentrations below the critical micelle concentration (CMC), LPLs can increase the dimerization constant for membrane-bound gramicidin up to 500-fold (at 2 microM). The relative potency increases as a function of the size of the polar head group, but does not seem to vary as a function of head group charge. The increased dimerization constant results primarily from an increase in the rate constant for channel formation, which can increase more than 100-fold (in the presence of LPC and LPI), whereas the channel dissociation rate constant decreases only about fivefold. The LPL effect cannot be ascribed to an increased membrane fluidity, which would give rise to an increased channel dissociation rate constant. The ability of LPC to decrease the channel dissociation rate constant varies as a function of channel length (which is always less than the membrane's equilibrium thickness): as the channel length is decreased, the potency of LPC is increased. LPC has no effect on membrane thickness or the surface tension of monolayers at the air/electrolyte interface. The bilayer-forming glycerolmonooleate does not decrease the channel dissociation rate constant. These results show that LPLs alter gramicidin channel function by altering the membrane deformation energy, and that the changes in deformation energy can be related to the molecular "shape" of the membrane-modifying compounds. Similar alterations in the mechanical properties of biological membranes may form a general mechanism by which one can alter membrane protein function.     

Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Exceptionally high-affinity binding in A7r5 and A10 cells

Calcium channel currents were studied in the A10 and A7r5 cell lines derived from rat thoracic aorta muscle cells. The whole-cell variation of the patch voltage clamp technique was used. Results with each cell line were nearly identical. Two types of Ca channels were found in each cell line that are similar to the L-type and T-type Ca channels found in excitable cells. Nimodipine block of the L-type Ca channels in both cell lines is more potent than in previously studied tissues. The kinetics of nimodipine block are accounted for by a model that postulates 1:1 drug binding to open Ca channels with an apparent dissociation constant (KO) of 16-45 pM. In A7r5 cells, the rate of onset of nimodipine block increases with the test potential, in quantitative agreement with the model of open channel block. The apparent association rate (f) is 1.4 x 10(9) M-1 s-1; the dissociation rate (b) is about 0.024 s-1. In anterior pituitary cells (GH4C1 cells), KO is 30 times larger; b is only twice as fast, but f is 15 times slower. The comparative kinetic analysis indicates that the high-affinity binding site for nimodipine is similar in both GH4C1 and A7r5 cells, but nimodipine diffuses much faster or has a larger partition coefficient into the plasmalemma of A7r5 cells than for GH4C1 cells. Unusually high-affinity binding was not observed in earlier 45Ca flux studies with A10 and A7r5 cells. The model of open channel block accounts for the discrepancy; only a small fraction of the Ca channels are in the high affinity open state under the conditions used in 45Ca flux studies, so an effective binding constant is measured that is much greater than the dissociation constant for high-affinity binding.     

Towards a mechanism of function of the viral ion channel Vpu from HIV-1

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo-oligomeric Vpu bundles. Reconstitution of both full length Vpu(1-81) and a short, the transmembrane (TM) domain comprising peptide Vpu(1-32) into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.     

Kinetics of the actions of caffeine and procaine on the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles

The effects of caffeine and procaine on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx in SR vesicles was measured by following the change in light scattering intensity using a stopped flow apparatus. From the kinetic analysis of the rate of choline influx, the following results were obtained. (1) The rate of choline influx was enhanced when Ca2+ bound to the Ca2+-receptor site of the Ca2+-gated cation channel. (2) Caffeine enhanced the choline influx by increasing only the affinity of Ca2+ for the receptor site of the channel and thus regulated the equilibrium between open and closed states of the channel. The affinity increased about 14-fold upon caffeine binding. The dissociation constant of caffeine was 10 mM. (3) In contrast, procaine itself blocked the choline influx mediated by the Ca2+-gated cation channel. The blockade followed a single-site titration curve with a Ca2+-dependent dissociation constant of 0.44 mM at 2 x 10(-6) M Ca2+. The Ca2+-dependence was explained by assuming that procaine would bind to the inhibitory site only when the channel was open. (4) Procaine also inhibited the choline influx enhanced by caffeine. The blockade could be explained on the basis of the above kinetic model.     

Block of wild-type and inactivation-deficient cardiac sodium channels IFM/QQQ stably expressed in mammalian cells

The role of inactivation as a central mechanism in blockade of the cardiac Na(+) channel by antiarrhythmic drugs remains uncertain. We have used whole-cell and single channel recordings to examine the block of wild-type and inactivation-deficient mutant cardiac Na(+) channels, IFM/QQQ, stably expressed in HEK-293 cells. We studied the open-channel blockers disopyramide and flecainide, and the lidocaine derivative RAD-243. All three drugs blocked the wild-type Na(+) channel in a use-dependent manner. There was no use-dependent block of IFM/QQQ mutant channels with trains of 20 40-ms pulses at 150-ms interpulse intervals during disopyramide exposure. Flecainide and RAD-243 retained their use-dependent blocking action and accelerated macroscopic current relaxation. All three drugs reduced the mean open time of single channels and increased the probability of their failure to open. From the abbreviation of the mean open times, we estimated association rates of approximately 10(6)/M/s for the three drugs. Reducing the burst duration contributed to the acceleration of macroscopic current relaxation during exposure to flecainide and RAD-243. The qualitative differences in use-dependent block appear to be the result of differences in drug dissociation rate. The inactivation gate may play a trapping role during exposure to some sodium channel blocking drugs.     

How ATP inhibits the open K(ATP) channel

ATP-sensitive potassium (K(ATP)) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 microM.     

Curare binding and the curare-induced subconductance state of the acetylcholine receptor channel.

The curare-induced subconductance state of the nicotinic acetylcholine receptor (AChR) of mouse skeletal muscle was examined using the patch-clamp technique. Two mechanisms for the generation of subconductance states were considered. One of these mechanisms entails allosteric induction of a distinct channel conformation through the binding of curare to the agonist binding site. The other mechanism entails the binding of curare to a different site on the protein. Occupation of this site would then limit the flow of ions through the channel. The voltage dependence and concentration dependence of subconductance state kinetics are consistent with curare binding to a site within the channel. The first order rate constant for binding is 1.2 X 10(6) M-1s-1 at 0 mV, and increases e-fold per 118 mV of membrane hyperpolarization. The rate of curare dissociation from this site is 1.9 X 10(2)s-1 at 0 mV, and decreases e-fold per 95 mV hyperpolarization. The equilibrium constant is 1.4 X 10(-4) M at 0 mV, and decreases e-fold per 55 mV hyperpolarization. This voltage dependence suggests that the fraction of the transmembrane potential traversed by curare in binding to this site is 0.46 or 0.23, depending on whether one assumes that one or both charges of curare sense the electric field. Successive reduction and alkylation of the AChR agonist binding sites with dithiothreitol (DTT) and N-ethyl maleimide (NEM), a treatment which results in the loss of responsiveness of the AChR to agonists, produced no change in curare-induced subconductance events, despite the fact that after this treatment most of the channel openings occurred spontaneously. Mixtures of high concentrations of carbamylcholine (CCh) with a low concentration of curare, which produce channel openings gated predominantly by CCH, resulted in subconductance state kinetics similar to those seen in curare alone at the same concentration. Thus displacement by CCh of curare from the agonist binding sites does not prevent curare from inducing subconductances. The results presented here support the hypothesis that curare induces subconductance states by binding to a site on the receptor other than the agonist binding sites, possibly within the channel pore. It is the occupation of this site by curare that limits the flow of ions through an otherwise fully opened channel.     

The multiple phosphorylation of the microtubule-associated protein MAP2 controls the MAP2:tubulin interaction

Pre-phosphorylation of the microtubule-associated protein MAP2 with the co-purifying cAMP-independent protein kinase (a) decrease the affinity of MAP2 for taxol-stabilised microtubules, (b) increases the dissociation rate constant for microtubule polymerisation, each of which is dependent upon the level of phosphorylation, but (c) has no effect on the association rate constant. Microtubule assembly has no effect on the kinetics of phosphorylation, whereas phosphorylation of pre-assembled microtubules causes their immediate depolymerisation at a rate which is proportional to the initial rate of phosphorylation. The results suggest that the modulated phosphorylation of MAP2 may regulate microtubule length in vivo.     

Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.