Markus Hanner

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.

High affinity of sigma1‐binding sites for sterol isomerization inhibitors: evidence for a pharmacological relationship with the yeast sterol C8–C7 isomerase

The sigma‐drug binding site of guinea‐pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8–C7 isomerase (ERG2 protein). Pharmacologically ‐ but not structurally ‐ the sigma1‐site is also related to the emopamil binding protein, the mammalian sterol C8–C7 isomerase. We therefore investigated if sterol C8–C7 isomerase inhibitors are high affinity ligands for the (+)‐[3H]‐pentazocine labelled sigma1‐binding site. Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma1‐binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY‐9944 (Ki 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM). Except for tamoxifen these affinities are essentially identical with those for the [3H]‐ifenprodil labelled sterol C8–C7 isomerase of S. cerevisiae. This demonstrates that sigma1‐binding protein and yeast isomerase are not only structurally but also pharmacologically related. Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma1‐binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis.

The beta subunit of the high conductance calcium-activated potassium channel. Identification of residues involved in charybdotoxin binding.

Coexpression of α and β subunits of the high conductance Ca2+-activated K+(maxi-K) channel leads to a 50-fold increase in the affinity for125I-charybdotoxin (125I-ChTX) as compared with when the α subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munujos, P., Knaus, H.-G., Kaczorowski, G. J., and Garcia, M. L. (1997) Proc. Natl. Acad. Sci. U. S. A.94, 2853–2858). To identify those residues in the β subunit that are responsible for this change in binding affinity, Ala scanning mutagenesis was carried out along the extracellular loop of β, and the resulting effects on 125I-ChTX binding were determined after coexpression with the α subunit. Mutagenesis of each of the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the extracellular loop of β was demonstrated after comparison of reactivities of native β and single-Cys-mutated subunits toN-biotin-maleimide. Negatively charged residues in the loop of β, when mutated individually or in combinations, had no effect on toxin binding with the exception of Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90, Tyr91, Thr93, and Glu94 are critical in conferring high affinity125I-ChTX binding to the α·β subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association and dissociation, but they do not alter the physical interaction of β with the α subunit. All these data, taken together, suggest that the large extracellular loop of the maxi-K channel β subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate conformation within the α subunit that allows high affinity ChTX binding.

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.

Scorpion toxins: Tools for studying K+ channels

Over the last period of time, a large number of scorpion toxins have been characterized. These peptidyl inhibitors of K+ channels have been very useful as probes for determining the molecular architecture of these channels, for purifying channels from native tissue and determining their subunit composition, for developing the pharmacology of K+ channels, and for determining the physiologic role that K+ channels play in target tissues. The large knowledge that we have developed regarding K+ channel function would not have been possible without the discovery of these peptidyl inhibitors. It is expected that as more novel peptides are discovered, our understanding of K+ channel structure and function will be further enhanced.

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.

Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus. The molecular weight of the native enzyme was about 320,000, as calculated by gel-filtration studies, and a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.

Purification and functional reconstitution of high-conductance calcium-activated potassium channel from smooth muscle.

Publisher Summary High-conductance Ca2+-activated K+ (maxi-K+) channels are activated by both membrane depolarization and binding of Ca 2+ to sites at the intracellular face of the channel. Maxi-K+ channels are present in both electrically excitable and nonexcitable cells, and display high conductance and selectivity for K+. These channels are involved in regulation of the excitation-contraction coupling process in smooth muscle, as well as in control of transmitter release from neuroendocrine tissues. The pharmacology of maxi-K+ channels has been developed during the last few years and efforts are continuing to identify novel and selective modulators of this channel family. This chapter discusses procedures developed to purify maxi-K+ channels from smooth muscle tissues, as well as the way purified preparation can be reconstituted into liposomes for determination of channel activity. The chapter also discusses ways of obtaining protein sequence information from components of the maxi-K+ channel preparation that are useful for obtaining full-length cDNA clones of these proteins.

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).