Chemical tools for K(+) channel biology

Abstract

K(+) channels are revered for their universal action of suppressing electrical activity in nerve and muscle, as well as regulating salt and water transport in epithelial tissues involved in metabolism and digestion. These multisubunit membrane-embedded proteins carry out their physiological chore, selectively allowing the passage of potassium across the membrane, in response to changes in membrane voltage and ligand concentration. Elucidating the diverse gating properties of K(+) channels is of great biological interest since their molecular motions provide insight into how these structurally similar proteins function in a wide variety of tissues. Armed with patch clamps, chart recorders, and now high-resolution structures, electrophysiologists have been dipping into the top tray of the chemist's tool box: synthesizing cysteine-modifying agents and organic cations and grinding up insects, spiders, and other vermin to isolate natural products to poke, probe, and prod K(+) channels. Recently, there has been further cross-fertilization between chemists and K(+) channelologists, resulting in greater accessibility to more elaborate synthetic methodologies and screening approaches. In this review, we catalogue the evolution of chemical tools and approaches that have been utilized to elucidate the mechanistic underpinnings of K(+) channel biology.

Figure 1. Structural models of inward rectifier Kir and voltage-gated Kv K⁺ channels

High resolution structures of the (A) Kirbac3.1 and (B) Kv2.1 paddle chimera K⁺ channels, respectively (Protein Data Bank codes 1X6L and 2R9R). For clarity, the front and back subunits have been removed. For Kirbac3.1, the gray and yellow colors demark the lipid- and pore-facing residues determined by a mutagenic yeast screen; orange and red spacefill atoms are the pairs of intrasubunit interacting residues determined by second site suppressor mutants. For Kv2.1, red color identifies the positively charged S4 helix; pink residues have been morphed into aromatic groups to frame the extracellular tetraethylammonium (TEA) binding site; red TEA molecule shows the extracellular binding site; blue TEA molecule: internal binding site. Purple spheres denote K⁺ ions in the selectivity filter for both structures.

Figure 2. Tethered blockers as molecular calipers

(A) At low reagent concentration, tethered blockers first bind to the channel pore and then react with the target nucleophile. Cartoon depicts nucleophile 1 (Nu¹) reacting with the electrophile on the tethered blocker whereas the linker of tethered blocker is too short to permit reaction with nucleophile 2 (Nu²). Upon modification with a tethered blocker, subsequent reactions with unreacted nucleophiles are inhibited by the covalently-tethered blocker. (B) Chemical components of tethered blockers (listed from top to bottom). Blockers: tetraethylammonium (TEA); charybdotoxin (CTX). Linkers: polyglycine (synthetic control of tether length); bis(N-phenylcarbamoyl)disulfane (cleavable by reductant); azobenzene (light-controllable). Electrophiles: maleimide (thiol specific); acrylamide, chloroacetamide, epoxide (non-specific).

Figure 3. Unnatural amino acid incorporation into K⁺ channels

(A) In vivo nonsense suppression in Xenopus laevis oocytes. An appropriately protected (NVOC), cyanomethyl ester-activated unnatural amino acid is coupled to either the 2′ or 3′ hydroxyl of dinucleotide pdCpA. This product, shown here with the unnatural amino acid (UUA) as a red asterisk, is ligated enzymatically to a truncated, amber suppressor tRNA from Tetrahymena thermophilia. The full-length, aminoacylated tRNA and the UAG-mutated K⁺ channel mRNA are co-injected into oocytes. UAG denotes the site of UAA incorporation. K⁺ channel function is measured using two-electrode voltage clamp. (B) Examples of UAA incorporated into K⁺ channels. Top, photocleavable (Npg) and fluorescent (Aladan); Bottom, fluorophenylalanines. The effect of serial fluorination is shown with color images of the 6–31G** electrostatic potential with red and blue corresponding to −20 and 20 kcal/mol, respectively. (C) UAA incorporation in mammalian cells via DNA transfection of cDNA plasmids carrying co-evolved tRNA/synthetase pairs and K⁺ channel cDNA with a TAG mutation. Successful UAA incorporation requires the prolonged exposure to the free UAA (red asterisks). K⁺ channel function is assayed by electrophysiological (patch-clamp) approaches. (D) Schematic of the expressed protein ligation (EPL) method as applied to K⁺ channels. UAA containing K⁺ channel complexes can be probed biochemically, functionally in lipid bilayers and by structural methods.