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 approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.
Highlights
Members of the Kir family of potassium channels are regulated by many different intracellular ligands
Charged residues within the proximal C terminus are known to be important for binding phosphoinositides, and an aspartate within this region of GIRK2 and GIRK4 mediates the activation of GIRK channels by Naϩ (7) and inhibition by eicosanoids (22)
Mutation of Cytoplasmic Cysteines within GIRK1 and GIRK2 Produces Substantial Changes in Functional Expression—In inside-out membrane patches from Xenopus oocytes, GIRK1/ GIRK2 channel activity was maintained by the inclusion of ATP in the bath solution, albeit at a lower level than basal cell-attached activity
Summary
CDNA Clones and Construction of Point Mutants—Rat GIRK1 and GIRK2 cDNAs were subcloned into the BamHI and EcoRI site of the pBG7.2 vector (8) and into similar sites of the pGEMHE vector. The point mutations to GIRK1 and GIRK2 were performed in the pBG7.2 vector using the GeneEditorTM in vitro site-directed mutagenesis system (Promega). Electrophysiology—Two-electrode voltage clamp recordings were performed with an OC-725B amplifier (Warner Instruments Corp., Hamden, CT) as described in Ho and Murrell-Lagnado (8). The holding potential was Ϫ80 mV, and records were filtered at 1 kHz. Patch clamp recordings were made using an AXOPATCH 200A amplifier (Axon Instruments) as described in Ho and Murrell-Lagnado (8). Pipettes filled with pipette solution (mM) (96 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, pH 7.2, with KOH) had resistances between 0.5 and 3 megohms. The values are presented as means Ϯ S.D. Single channel data were analyzed using TAC software to determine unitary current amplitudes and open time distributions (version 2.51, SKALAR Instruments, Seattle, WA). Levels of statistical significance were: * ϭ p Ͻ 0.001 and ** ϭ p Ͻ 0.05
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