A Role for the Middle C Terminus of G-protein-activated Inward Rectifier Potassium Channels in Regulating Gating

Abstract

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(؊)) increased the current by ϳ17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(؉)), abolished the current. There was no effect on single-channel conductance. Members of the Kir family of potassium channels are regulated by many different intracellular ligands. For the GIRK 1 channels, it has been shown that G ␤␥ subunits (1-3), phosphoinositides (4 -6), and Na ϩ (7-9) all directly interact with the channel to increase activity. In contrast, polyamines and Mg 2ϩ interact with the channel to inhibit currents by a mechanism of pore blockade (10 -12). The majority of regulators of Kir channels bind to regions within the cytoplasmic N-and C-terminal tails of the Kir subunits. Kir subunits contain two transmembrane (TM) segments with a short N terminus and a long C terminus of between 200 and 300 amino acids. Little is known of the structure of the cytoplasmic regions or of the conformational changes that occur during the process of regulation.Kir subunits assemble as tetramers, and the second TM helices line the pore and form a gate that controls ion flux (13-17). For different members of the Kir family, the region of high sequence similarity extends ϳ30 amino acids from TM1 into the N terminus and ϳ170 amino acids from TM2 into the C terminus. The cytoplasmic regions adjacent to TM1 and TM2 have been proposed to form an intracellular vestibule of the pore (18). Modification of cysteines introduced into these regions of Kir2.1 inhibit channel currents, and at the E224C position the extent of inhibition was shown to be proportional to the size of the modifying reagent, suggesting that the mechanism was one of pore blockade. The Glu-224 residue in Kir2.1 and a histidine at a similar position in Kir6.2 (His-216) were also shown to affect polyamine block by electrostatic interactions, suggesting that they face the permeation pathway (19 -21). Positively charged residues within the proximal C terminus are also known to be important for binding phosphoinositides, and an aspartate within this region of...

L-DOPA-quinone Mediated Recovery from GIRK Channel Firing Inhibition in Dopaminergic Neurons

“…28 The alkylation by sulfhydryl-modifying reagents of Nterminal cysteine 65 (C65) and C-terminal cysteine 321 (C321) residues, both placed within the cytoplasmic domain of GIRK2 channel, determine the inhibition of GIRK mediated current in GIRK1/GIRK2 heteromeric channels. 29 In particular, the alkylation of C65 was shown to have a high impact in current modulation, and it was found to be considerably more accessible than C321 to sulfhydryl modifying reagents, as is also demonstrated by the X-ray crystallographic structure of murine GIRK2 channel (98.3% identity with human isoform) in the preopen conformation, once complexed with β−γ G-protein subunits, dioctanoyl-l-alpha-phosphatidyl-D-myo-inositol-4,5diphosphate (PIO), and Na + (PDB code 4KFM). For this reason, we assumed C65 as the specific cysteine residue to which DOPA-quinone would bind.…”

Dual activation of neuronal G protein-gated inwardly rectifying potassium (GIRK) channels by cholesterol and alcohol

“…1b). Subsequent addition of a small molecular inhibitor of GIRK2 channels, MTS-HE (100 μM) 37 , abruptly slowed the rate of quenching, indicating closure of GIRK2 channels (‘GIRK2/PIP 2  + HE’, Fig. 1b).…”

Dynamic role of the tether helix in PIP2-dependent gating of a G protein–gated potassium channel

“…To test this idea, we used a strategy of chemical modification in which a single cysteine is introduced at 5′K or 6′K and is then probed with a membrane permeant MTS compound. This technique has the advantage of enabling the study of a channel before and after modification and has been used previously to probe gating structures of inwardly rectifying potassium channels ( Guo et al, 2002 ; Lopes et al, 2002 ; Xiao et al, 2003 ; Bodhinathan and Slesinger, 2013 ). Here, we focused on the effect of MTS hydroxyethyl (HE), which would covalently attach a CH 2 -CH 2 -OH side chain to the sulfhydryl of the engineered cysteine, mimicking a serine/threonine type of amino acid substitution, i.e., a polar, uncharged residue.…”