Pharmacology of voltage-gated and calcium-activated potassium channels.

Several important new findings have furthered the development of voltage-gated and calcium-activated potassium channel pharmacology. The molecular constituents of several members of these large ion channel families were identified. Small-molecule modulators of some of these channels were reported, including correolide, the first potent, small-molecule, natural product inhibitor of the Shaker family of voltage-gated potassium channels to be disclosed. The initial crystal structure of a bacterial potassium channel was determined; this work gives a physical basis for understanding the mechanisms of ion selectivity and ion conduction. With the recent molecular characterization of a potassium channel structure and the discovery of new templates for channel modulatory agents, the ability to rationally identify and develop potassium channel agonists and antagonists may become a reality in the near future.     

Structural and functional modularity of voltage-gated potassium channels

Sequence similarity among known potassium channels indicates the voltage-gated potassium channels consist of two modules: the N-terminal portion of the channel up to and including transmembrane segment S4, called in this paper the 'sensor' module, and the C-terminal portion from transmembrane segment S5 onwards, called the 'pore' module. We investigated the functional role of these modules by constructing chimeric channels which combine the 'sensor' from one native voltage-gated channel, mKv1.1, with the 'pore' from another, Shaker H4, and vice versa. Functional studies of the wild type and chimeric channels show that these modules can operate outside their native context. Each channel has a unique conductance-voltage relation. Channels incorporating the mKv1.1 sensor module have similar rates of activation while channels having the Shaker pore module show similar rates of deactivation. This observation suggests the mKv1.1 sensor module limits activation and the Shaker pore module determines deactivation. We propose a model that explains the observed equilibrium and kinetic properties of the chimeric constructs in terms of the characteristics of the native modules and a novel type of intrasubunit cooperativity. The properties ascribed to the modules are the same whether the modules function in their native context or have been assembled into a chimera.     

Structural basis for toxin resistance of beta4-associated calcium-activated potassium (BK) channels

The functional diversity of large conductance Ca(2+)- and voltage-dependent K(+) (BK) channels arises mainly from co-assembly of the pore-forming mSlo alpha subunits with four tissue-enriched auxiliary beta subunits. The structural basis of the interaction between alpha subunits with beta subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hbeta4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K(d) = 17 nm, suggesting that the hbeta4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the alpha subunits that could "permanently" block 15% of mSlo+hbeta4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between alpha and beta subunits and will improve the design of channel inhibitors.     

Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.     

Subconductance states of single sodium channels modified by chloramine-T and sea anemone toxin in neuroblastoma cells

Single channel currents of chloramine-T (Chl-T) and sea anemone toxin (ATX-II) modified sodium channels were studied in neuroblastoma cells. With both substances similar subconductance states have been observed. The conductances of the sublevels were multiples of the unit step which was about onefourth of the most frequently occurring main conductance. Thus, the current levels observed were one fourth, half and five-fourths of the main current size. Both substances caused a slower decay of the averaged current compared to the current of the native channels. The main single-channel conductance was 15.2 pS (T=16°C) for the Chl-T and 10.8 pS (T=12°C) for the ATX-II modified channels. The channel open time was doubled by ATX-II, but was not increased significantly by Chl-T. The existence of the subconductance states suggests that the native channels may also have multiple open conformations.     

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.     

Interaction between sea anemone toxin BgTX8 and ionic channels of neurons isolated from mammals

The action of the toxin BgTX₈ separated from the sea actiniaBunodosoma granolifera on transient tetrodotoxin-sensitive sodium and outward potassium currents of units isolated from rat sensory ganglia was investigated using techniques of voltage clamping at the membrane and intracellular perfusion. It was found that BgTX₈ decelerates the inactivation kinetics but has little effect on activation kinetics of sodium current. At the same time, a 5–10% increase in the amplitude of inward current was often observed at holding potentials of about –100 to –120 mV at the membrane. The dissociation constant of the receptor-toxin equals 4×10^–6 M and is adequately described by Langmuir's isotherm. It was also established that intracellular perfusion of neurons with anemone toxin-containing solution leads to a reduction in the amplitude of sodium current and decelerates its inactivation process. Suppression of outward potassium current was also noted.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Brain Research, Academy of Sciences, Havana, Cuba. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 32–37, January–February, 1988.     

Preparation and characterization of a biologically active spin-labeled sea anemone toxin

A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin-labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over thepH range 2–9. The ESR data also show evidence for multimetric states of SL-AP-B over thepH range 2–9, with maximum aggregation at pH 4.5–5, and a slow disaggregation when thepH is adjusted to 8–9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.Abbreviations AP-A anthopleurin-A - AP-B anthopleurin-B - ATX Ia toxin Ia fromAnemonia sulcata - Sh I neurotoxin I fromStichodactyla helianthus - TFA trifluoroacetic acid - SL-AP-B AP-B labeled at the N-terminus with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide     

Binding of the sea anemone polypeptide BDS II to the voltage-gated sodium channel.

BDS II, a 43-residue polypeptide from the sea anemone Anemonia sulcata, is reported to have both antihypertensive and antiviral activity. This polypeptide possesses a number of sequence and structural similarities to a class of cardiotonic proteins which bind to receptor site 3 of the voltage-gated sodium channel. In contrast to these cardiostimulant proteins, which produce positive inotropic effects at concentrations of 2-15 nM, BDS II produced a weak negative inotropic effect upon isolated guinea-pig atria, with doses of 90 and 180 nM depressing contractile strength by 15 and 28%, respectively. BDS II also competed with a 125-iodine labelled derivative of AP-A (a representative of the cardiostimulant proteins) bound to sodium channels in rat brain synaptosomes. The IC50 for BDS II versus AP-A was 5.2 microM. BDS II may therefore be considered an antagonist for receptor site 3 of the voltage-gated sodium channel. Structural differences between BDS II and the agonist AP-A which may give rise to their different effects on the sodium channel are considered.     

Brownian dynamics simulations of the recognition of the scorpion toxin maurotoxin with the voltage-gated potassium ion channels

The recognition of the scorpion toxin maurotoxin (MTX) by the voltage-gated potassium (Kv1) channels, Kv1.1, Kv1.2, and Kv1.3, has been studied by means of Brownian dynamics (BD) simulations. All of the 35 available structures of MTX in the Protein Data Bank (http://www.rcsb.org/pdb) determined by nuclear magnetic resonance were considered during the simulations, which indicated that the conformation of MTX significantly affected both the recognition and the binding between MTX and the Kv1 channels. Comparing the top five highest-frequency structures of MTX binding to the Kv1 channels, we found that the Kv1.2 channel, with the highest docking frequencies and the lowest electrostatic interaction energies, was the most favorable for MTX binding, whereas Kv1.1 was intermediate, and Kv1.3 was the least favorable one. Among the 35 structures of MTX, the 10th structure docked into the binding site of the Kv1.2 channel with the highest probability and the most favorable electrostatic interactions. From the MTX-Kv1.2 binding model, we identified the critical residues for the recognition of these two proteins through triplet contact analyses. MTX locates around the extracellular mouth of the Kv1 channels, making contacts with its beta-sheets. Lys23, a conserved amino acid in the scorpion toxins, protrudes into the pore of the Kv1.2 channel and forms two hydrogen bonds with the conserved residues Gly401(D) and Tyr400(C) and one hydrophobic contact with Gly401(C) of the Kv1.2 channel. The critical triplet contacts for recognition between MTX and the Kv1.2 channel are Lys23(MTX)-Asp402(C)(Kv1), Lys27(MTX)-Asp378(D)(Kv1), and Lys30(MTX)-Asp402(A)(Kv1). In addition, six hydrogen-bonding interactions are formed between residues Lys23, Lys27, Lys30, and Tyr32 of MTX and residues Gly401, Tyr400, Asp402, Asp378, and Thr406 of Kv1.2. Many of them are formed by side chains of residues of MTX and backbone atoms of the Kv1.2 channel. Five hydrophobic contacts exist between residues Pro20, Lys23, Lys30 and Tyr32 of MTX and residues Asp402, Val404, Gly401, and Arg377 of the Kv1.2 channel. The simulation results are in agreement with the previous molecular biology experiments and explain the binding phenomena between MTX and Kv1 channels at the molecular level. The consistency between the results of the BD simulations and the experimental data indicated that our three-dimensional model of the MTX-Kv1.2 channel complex is reasonable and can be used in additional biological studies, such as rational design of novel therapeutic agents blocking the voltage-gated channels and in mutagenesis studies in both the toxins and the Kv1 channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp378 of the Kv1.2 channel is critical for its recognition and binding functionality toward MTX. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating this might be a new clue for additional functional study of Kv1 channels.     

Brownian dynamics simulations of the recognition of the scorpion toxin P05 with the small-conductance calcium-activated potassium channels

The recognition of the scorpion toxin P05 and the small-conductance, calcium-activated potassium (SK) channels, rsk1, rsk2, and rsk3, has been studied by means of the Brownian dynamics (BD) method. All of the 25 available structures of P05 in the RCSB Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformation of P05 affects both the recognition and binding between the two proteins significantly. Comparing the top four high-frequency structures of P05 binding to the SK channels, we found that the rsk2 channel, with high frequencies and lowest electrostatic interaction energies (E (int)(ES)), is the most favorable for P05 binding, while rsk3 is intermediate, and rsk1 is the least favorable. Among the 25 structures of P05, the 13th structure docks into the binding site of the rsk2 channel with the highest probability and most favorable electrostatic interactions. From the P05-rsk2 channel binding model, we identified the residues critical for the recognition of these two proteins through triplet contact analyses. P05 locates around the extracellular mouth of the SK channels and contacts the SK channels using its alpha-helix rather than beta-sheets. The critical triplet contacts for recognition between P05 and the rsk2 channel are Arg6 (P05)-Asp364 (SK), Arg7 (P05)-Asn368 (SK), and Arg13 (P05)-Asp341 (SK). The structure of the P05-rsk2 complex with the most favorable electrostatic interaction energy was further refined by molecular mechanics, showing that six hydrogen bonding interactions exist between P05 and the rsk2 channel: one hydrogen bond is formed between Arg6 (P05) and Asp364(D) (rsk2); Arg7 (P05) forms three hydrogen bonds with Asp341(B) (rsk2)) and Asp364(C) (rsk2); two hydrogen bonds are formed by Arg13 (P05) with Asp341(A) (rsk2) and Asp364(B) (rsk2). The simulation results are in good agreement with the previous molecular biological experiments and can explain the binding phenomena between P05 and SK channels at the level of molecular structure. The consistency between the results of the BD simulations and the experimental data indicated that our 3D model of the P05-rsk2 channel complex is reasonable and can be employed in further biological studies, such as rational design of the novel therapeutic agents blocking the small-conductance, calcium-activated and apamin-sensitive potassium channels, and for mutagenesis studies in both toxins and SK channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp364 of the rsk2 channel is critical for its recognition and binding functionality towards P05. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating that this might be a new clue for further functional study of SK channels.     

Conditional and unconditional inhibition of calcium-activated potassium channels by reversible protein phosphorylation

Large conductance, calcium-activated potassium channels (BK(Ca) or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulation of native BK(Ca) channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GH(4)C(1)). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BK(Ca) channel activity approximately 3-fold. Dephosphorylated channels could be activated fully by physiological increases in cytoplasmic calcium or membrane depolarization. In contrast, endogenous protein kinases inhibited BK(Ca) channel activity at two functionally distinct sites. A closely associated, cAMP-dependent protein kinase rapidly reduced channel activity in a conditional manner that could be overcome completely by increasing cytoplasmic free calcium 3-fold or 20 mV further depolarization. Phosphorylation at a pharmacologically distinct site inhibited channel activity unconditionally by reducing availability to approximately half that of maximum at all physiological calcium and voltages. Conditional versus unconditional inhibition of BK(Ca) channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.     

Atomic scale structure and functional models of voltage-gated potassium channels.

Recent mutagenesis experiments have confirmed our hypothesis that a segment between S5 and S6 forms the ion selective portion of voltage-gated ion channels. Based on these and other new data, we have revised previous models of the general folding pattern of voltage-gated channel proteins and have developed atomic scale models of the entire transmembrane region of the Shaker A K+ channel. In these models, the ion selective region is a beta-barrel that spans the outer half of the membrane. The inner half of the pore is larger. The voltage-dependent conformational changes of activation gating are modeled to occur by the "helical screw" mechanism, in which the four S4 segments move along and rotate about their axes. These changes are followed by a voltage-independent conformational change, in which the segments linking S4 to S5 move from blocking the intracellular entrance of the pore to forming part of the lining of the large inner portion of the pore. The NH2-terminal of the protein was modeled as an alpha-helix that plugs the intracellular half of the pore to inactivate the channel.     

Ionic channels induced by sea nettle toxin in the nodal membrane.

Toxin isolated from nematocysts of the sea nettle Chrysaora quinquecirrha (SNTX) is known to depolarize nerve and muscle membranes and to increase the miniature end-plate potentials' frequency. To investigate its mode of action at the membrane level, we have studied the toxin's effects on the frog myelinated nerve fibre. We show that SNTX creates large cation-selective channels that open and close spontaneously. The conductance of these channels, almost constant in the voltage range - 100 to + 50 mV, is 760 pS. The SNTX-induced channels are almost equally permeable to Na+, Li+, K+, and Cs+, but are impermeable to Ca++. The open and closed times of SNTX-induced channels are voltage dependent, the open probability increasing with increased negative membrane potentials. To our knowledge, this is the first demonstration of the production of single-channel currents by a toxin, in a biological membrane.     

Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels

An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.     

Functional role of a conserved aspartate in the external mouth of voltage-gated potassium channels.

Mutation of the glycines in a conserved Gly-Tyr-Gly-Asp sequence in the P-region of voltage-gated K channels has identified determinants of Na/K selectivity. But the function of the negatively charged Asp is not known because mutations at this position are not tolerated, owing to the fourfold replication of mutations in a tetrameric channel. We have successfully mutated Asp378-->Thr in a tandem dimer Kv2.1 construct to yield a twofold neutralization of charge at this site. When expressed in Xenopus oocytes, the mutated channels showed markedly altered ion conduction and blockade. Potassium conduction in the inward direction was selectively reduced, so that the instantaneous current-voltage relationship obtained in isotonic KCl became strongly outwardly rectifying. The relative permeability to Na+, PNa/PK, increased from 0.02 to 0.10 without changing the ion selectivity sequence K > Rb >> Cs >> Na. The IC50 for block by external tetraethylammonium (TEA) increased more than 100-fold without affecting block by internal TEA. We conclude that Asp378 is an essential part of a potassium ion binding site associated with the Na/K selectivity filter at the external mouth of the pore.