Robert S. Slaughter

PHARMACOLOGY OF POTASSIUM CHANNELS

Publisher Summary Potassium channels represent the largest and most diverse family of ion channels. K + channels can be divided into two groups, voltage-gated and ligand-gated channels, depending on the stimulus that triggers the conformational changes leading to channel opening. K + channels share in common the feature of having high selectivity for K + as the permeating ion. Because of this property, and given the wide tissue distribution of these proteins, K + channels have been postulated to be involved in a variety of physiologic processes, such as control of cell excitability, release of neurotransmitters, secretion of hormones, regulation of fluid secretion, and clonal expansion of cells of the immune system. This chapter mentions the nature and properties of the specific channels those are present. A large number of voltage-dependent K + channels are known to exist. They are presumed to contain six α-helical transmembrane domains (S1-S6) with a segment between S5 and S6, termed the P region that contributes to the channels pore. The P region is the most conserved domain among all different types of K + channels and, because it is not large enough to cross the membrane in an α-helical conformation, it has been proposed to form a p-hairpin-like structure. Given the fact that some peptidyl blockers display a broad spectrum of interaction with different family members, this review is divided into three major areas; voltage-gated K + channels, Ca 2+ - activated K + channels, and ATP-dependent K + channels. Discussed are the peptidyl blockers derived and the peptidyl inhibitors isolated from scorpion venoms, the peptidyl blockers from sea anemone and the spider venom and nonpeptidyl blockers. Ca 2+ -activated K + channels are discussed; including the interaction of the peptide ChTX with maxi-K channels and the nonpeptidyl maxi-K channel modulators. Also discussed are the small-conductance Ca 2+ -activated K + channels and the ATP-dependent K + channels.

Complex Subunit Assembly of Neuronal Voltage-gated K+Channels BASIS FOR HIGH-AFFINITY TOXIN INTERACTIONS AND PHARMACOLOGY

Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+(Kv1) channels in rat cerebellum that are labeled by125I-margatoxin (125I-MgTX;K d , 0.08 pm). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1.1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (<20%) seem to be homotetrameric Kv1.2 channels. Heterologous coexpression of Kv1.1 and Kv1.2 subunits in COS-1 cells leads to the formation of a complex that combines the pharmacological profile of both parent subunits, reconstituting the native MgTX receptor phenotype. Subunit assembly provides the structural basis for toxin binding pharmacology and can lead to the association of as many as three distinct channel subunits to form functional K+channels in vivo.

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes.

The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.

Binding of Correolide to Kv1 Family Potassium Channels MAPPING THE DOMAINS OF HIGH AFFINITY INTERACTION

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, Kv1.3, and [3H]dihydrocorreolide (diTC) binds with high affinity (K d ∼ 10 nm) to membranes from Chinese hamster ovary cells that express Kv1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F., Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922–4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, Kv3.2. Transferring the pore from Kv1.3 to Kv3.2 produces a chimera that binds peptidyl inhibitors of Kv1.3 with high affinity, but not diTC. Transfer of the S5 region of Kv1.3 to Kv3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with Kv1.3, whereas transfer of the entire S5-S6 domain results in a normal Kv1.3 phenotype. Substitutions in S5-S6 of Kv1.3 with nonconserved residues from Kv3.2 has identified two positions in S5 and one in S6 that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to Kv3.2 after substitution of these three residues with the corresponding amino acids found in Kv1.3. These results suggest that lack of sensitivity of Kv3.2 to diTC is a consequence of the presence of Phe382 and Ile387 in S5, and Met458 in S6. Inspection of Kv1.1–1.6 channels indicates that they all possess identical S5 and S6domains. As expected, diTC binds with high affinity (K d values 7–21 nm) to each of these homotetrameric channels. However, the kinetics of binding are fastest with Kv1.3 and Kv1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify Kv1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.

Benzamide derivatives as blockers of Kv1.3 ion channel

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.

SCORPION TOXINS AS TOOLS FOR STUDYING POTASSIUM CHANNELS

The search for peptidyl inhibitors of K+ channels is a very active area of investigation. In addition to scorpion venoms, other venom sources have been investigated; all of these sources have yielded novel peptides with interesting properties. For instance, spider venoms have provided peptides that block other families of K+ channels (e.g., Kv2 and Kv4) that act via mechanisms which modify the gating properties of these channels. Such inhibitors bind to a receptor on the channel that is different from the pore region in which the peptides discussed in this chapter bind. In fact, it is possible to have a channel occupied simultaneously by both inhibitor types. It is expected that many of the methodologies concerning peptidyl inhibitors from scorpion venom, which have been developed in the past and outlined above, will be extended to the new families of K+ channel blockers currently under development.

Potent nor-triterpenoid blockers of the voltage-gated potassium channel Kv1.3 from Spachea correae

Abstract The isolation and structure elucidation of two novel nor-triterpenoid K V 1.3 potassium channel blockers correolide and dehydrocorreolide from the Costa Rican tree Spachea correae are reported.

Diterpenoid pyrones, novel blockers of the voltage-gated potassium channel Kv1.3 from fungal fermentations

Abstract The isolation, structure elucidation and chemical modification of nalanthalide, a novel diterpenoid pyrone blocker of the voltage-gated potassium channel Kv1.3 are reported. The structure–activity relationship of the derivatives with respect to various associated biological activities is also discussed.

Scorpion toxins and potassium channels.

Potassium channels are a group of proteins that have in common the property of selectively allowing the movement of K+ through aqueous pores in the membrane. Gating of these proteins occurs through conformational changes that are controlled by voltage and/or ligand binding. Accordingly, K+ channels can be divided into voltage-dependent and ligand-activated channels. These channels are involved in the control of cell resting potential, and in modulation of the electrical excitability of neuronal, endocrine, and muscle cells. A large amount of information regarding the structure and function of these proteins has become available in the last few years due to two major developments: (i) the molecular cloning of complementary DNAs (cDNAs) encoding these proteins, and (ii) the discovery of high-affinity peptidyl inhibitors in the venom of different species. These peptidyl inhibitors have been useful in the development of the pharmacology of K+ channels (Kaczorowski et al., 1996), and in determining the physiologic role that a particular K+ channel plays in a given cell or tissue (Garcia et al., 1995). Moreover, they have also been employed as a marker for channel purification from native tissues, thus allowing the determination of these channels’subunit composition (Garcia-Calvo et al., 1994;Giangiacomo et al., 1995; Parcej and Dolly, 1989; Rehm and Lazdunski, 1988). As a matter of fact, all auxiliary subunits of K+ channels, because of rheir well-understood mechanism of action, have allowed the identification and molecular characterization of thepore region of these channels(Gross ea al., 1994), and the determination of their sybunit stoichiometry (Mackinnon, 1991).

Characterization of Novel Probes of Voltage-Dependent Calcium Channels

Voltage-dependent Ca2+ channels have been identified in both electrically excitable and non-excitable cells. Several distinct types of Ca2+ channels have been detected in these preparations. Perhaps the best characterized channel, in terms of its pharmacological properties, is the L-type, or voltage-dependent, slowly inactivating, Ca2+ channel (PCa,L). This channel protein is a multi-drug receptor. Several different structural classes of molecules have been identified which bind in a potent fashion to unique receptor sites that are part of the channel. These agents modulate channel gating behavior. Originally, 3 high affinity drug binding sites were shown to be associated with PCa,L that is present in cardiac sarcolemmal membranes. These sites recognize dihydropyridine, aralkylamine, and benzothiazepine classes of organic Ca2+ entry blockers (for a review see Glossmann and Striessnig, 1988). By analyzing the binding of ligands which interact at these sites (e.g., through studies with [3H]nitrendipine, [3H]D-600, and [3H]diltiazem, respectively), it has been possible to demonstrate that 3 distinct receptors exist in a complex on PCa,L, and that these sites are allosterically coupled. From these data, a model was developed which describes the allosteric interactions between individual sites in the cardiac Ca2+ entry blocker receptor complex (Garcia et. al., 1986). Recently, a new structural class of organic Ca2+ channel inhibitor (i.e., certain substituted diphenylbutylpiperidines) has been identified and shown to bind to another unique site on the cardiac Ca2+ channel (King et. al., 1989).