Gregory J. Kaczorowski

Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release

We examined, using physiological and morphological techniques, the distribution of Ca(2+)-gated K+ (gKca) channels relative to the location of Ca2+ channels and transmitter release sites at the frog neuromuscular junction (NM). Charybdotoxin (ChTx) and iberiotoxin, blockers of gKca channels with large conductances, increase transmitter release at the frog NMJ. Intracellular Ca2+ buffers with rapid binding kinetics, dimethyl BAPTA and BAPTA, prevented the effect of ChTx, but EGTA, a Ca2+ buffer with similar affinity for Ca2+ but slower binding kinetics, did not. Dimethyl BAPTA and BAPTA, but not EGTA, caused a temporary increase in transmitter release. Labeling of gKca channels with ChTx-biotin revealed a series of bands located at the sites of Ca2+ channels, but this labeling did not occur in denervated preparations. Cross sections of NMJs revealed that gKca channels are clustered in the presynaptic membrane facing the postsynaptic membrane. We conclude that gKca channels are strategically clustered at the neurotransmitter release sites, where they can be quickly activated by Ca2+ entering the terminal.

High-conductance calcium-activated potassium channels; structure, pharmacology, and function.

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K+ channels. They are unique in their dual requirement for depolarization and Ca2+ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, α and Β. The α subunit is a member of theslo Ca2+-activated K+ channel gene family and forms the ion conduction pore. The Β subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against α and Β subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.

ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Voltage-gated sodium (NaV1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to NaV1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding NaV1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make NaV1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires NaV1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with NaV1.7 channels, inhibiting NaV1.7 with an IC50 value of 0.3 nM, compared with IC50 values of 30 to 150 nM for other heterologously expressed NaV1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Aβ-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II (125I-ProTx-II) binds with high affinity (Kd = 0.3 nM) to recombinant hNaV1.7 channels. Binding of 125I-ProTx-II is insensitive to the presence of other well characterized NaV1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the 125I-ProTx-II binding assay, described here, offers a new tool in the search for novel NaV1.7-selective blockers.

Use of toxins to study potassium channels

Potassium channels comprise groups of diverse proteins which can be distinguished according to each members biophysical properties. Some types of K+ channels are blocked with high affinity by specific peptidyl toxins. Three toxins, charybdotoxin, iberiotoxin, and noxiustoxin, which display a high degree of homology in their primary amino acid sequences, have been purified to homogeneity from scorpion venom. While charybdotoxin and noxiustoxin are known to inhibit more than one class of channel (i.e., several Ca2+-activated and voltage-dependent K+ channels), iberiotoxin appears to be a selective blocker of the high-conductance, Ca2+-activated K+ channel that is present in muscle and neuroendocrine tissue. A distinct class of small-conductance Ca2+-activated K+ channel is blocked by two other toxins, apamin and leiurotoxin-1, that share no sequence homology with each other. A family of homologous toxins, the dendrotoxins, have been purified from venom of various related species of snakes. These toxins inhibit several inactivating voltage-dependent K+ channels. Although molecular biology approaches have been employed to identify and characterize several species of voltagegated K+ channels, toxins directed against a particular channel can still be useful in defining the physiological role of that channel in a particular tissue. In addition, for those K+ channels which are not yet successfully probed by molecular biology techniques, toxins can be used as biochemical tools with which to purify the target protein of interest.

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.

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.

Ion Channels as Drug Targets: The Next GPCRs

Ion channels are well recognized as important therapeutic targets for treating a number of different pathophysiologies. Historically, however, development of drugs targeting this protein class has been difficult. Several challenges associated with molecular-based drug discovery include validation of new channel targets and identification of acceptable medicinal chemistry leads. Proof of concept approaches, focusing on combined molecular biological/pharmacological studies, have been successful. New, functional, high throughput screening (HTS) strategies developed to identify tractable lead structures, which typically are not abundant in small molecule libraries, have also yielded promising results. Automated cell-based HTS assays can be configured for many different types of ion channels using fluorescence methods to monitor either changes in membrane potential or intracellular calcium with high density format plate readers. New automated patch clamp technologies provide secondary screens to confirm the activity of hits at the channel level, to determine selectivity across ion channel superfamilies, and to provide insight into mechanism of action. The same primary and secondary assays effectively support medicinal chemistry lead development. Together, these methodologies, along with classical drug development practices, provide an opportunity to discover and optimize the activity of ion channel drug development candidates. A case study with voltage-gated sodium channels is presented to illustrate these principles.

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 Properties of the High-Affinity TRPV1 (VR1) Vanilloid Receptor Antagonist (4-Hydroxy-5-iodo-3-methoxyphenylacetate ester) Iodo-Resiniferatoxin

We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC50 = 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC50 = 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [3H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.

High-conductance calcium-activated potassium channels

Potassium channels represent a vast and diverse family of ion channel proteins which are found in many different types of tissues [1, 2]. These channels modulate the electrical excitability of certain cells, such as those derived from neuronal, endocrine and muscle sources. They also control the resting plasma membrane potential of a large variety of cells, regardless of whether the cells display electrically excitable or nonexcitable properties. K+ channels are routinely categorized according to their biophysical and pharmacological properties. However, these proteins may also be sub-divided into two major classifications depending on whether they are activated by changes in membrane potential (voltage-sensitive channels), or by an interaction with small molecule modulators (ligand-gated channels). Unfortunately, when compared with other types of ion channels, such as with the members of the voltage-gated Na+ and Ca2+ channel families [3], the biochemistry and molecular pharmacology of K+ channels is still rather undeveloped.