- Open Access
Structural changes in the cytoplasmic pore of the Kir1.1 channel during pHi-gating probed by FRET
© Lee and Shieh; licensee BioMed Central Ltd. 2009
- Received: 04 November 2008
- Accepted: 06 March 2009
- Published: 06 March 2009
Kir1.1 channels are important in maintaining K+ homeostasis in the kidney. Intracellular acidification reversibly closes the Kir1.1 channel and thus decreases K+ secretion. In this study, we used Foster resonance energy transfer (FRET) to determine whether the conformation of the cytoplasmic pore changes in response to intracellular pH (pHi)-gating in Kir1.1 channels fused with enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (ECFP-Kir1.1-EYFP). Because the fluorescence intensities of ECFP and EYFP were affected at pHi < 7.4 where pHi-gating occurs in the ECFP-Kir1.1-EYFP construct, we examined the FRET efficiencies of an ECFP-S219R-EYFP mutant, which is completed closed at pHi 7.4 and open at pHi 10.0. FRET efficiency was increased from 25% to 40% when the pHi was decreased from 10.0 to 7.4. These results suggest that the conformation of the cytoplasmic pore in the Kir1.1 channel changes in response to pHi gating such that the N- and C-termini move apart from each other at pHi 7.4, when the channel is open.
- HEK293T Cell
- Transmembrane Helix
- Hill Coefficient
- Membrane Patch
- Intracellular Acidification
K+ homeostasis is controlled by the secretion of K+ ions across the apical membrane of cortical collecting duct cells in the kidney. Low-conductance inwardly rectifying K+ channels are the channels primarily responsible for K+ secretion [1, 2]. These low-conductance K+ channels have been shown to be particularly sensitive to changes in the pHi. Intracellular acidification in the physiological range reversibly reduces the channel open probability and is thought to account for the subsequent decrease in K+ secretion [1–3]. Thus, the sensitivity of the apical K+ channel to the pHi is assumed to play a key role in K+ homeostasis.
The processes involved in the opening and closing of Kir1.1 channels in response to pHi changes are not completely understood. It has been suggested that the closure of the Kir1.1 pHi gate results from the occlusion of the tetrameric channel pore by the convergence of four leucines at the cytoplasmic apexes of the four inner transmembrane helices . In addition, it was recently proposed that H+ and PIP2 use a gating mechanism defined by conformational changes in the transmembrane helices and the selectivity filter and that the gating movement of the transmembrane helices is, in turn, controlled by an intrasubunit hydrogen bond between transmembrane domains 1 and 2 at the helix-bundle crossing .
It has been proposed that ligands gating Kir channels open or close the pore by initiating conformational changes in the cytoplasmic domains . The accessibility of N-terminal and C-terminal region cysteines C49 and C308 to methanethiosulfonate reagents has been shown to be pHi (state)-dependent, suggesting that pHi-gating may involve movements in the cytoplasmic-located pore, which is composed of both the N- and C-terminal regions . Furthermore, the interaction of the N- and C-termini has been suggested to be an important part of the channel gating mechanism . However, there is no direct evidence that the pHi can modulate the interaction of the N- and C-termini, either in vitro or in intact cells.
In this study, we used patch clamp fluorometry and Foster resonance energy transfer (FRET) microscopy to measure currents and probe the interactions of the N- and C-termini of the Kir1.1 (Kir1.1a) channel during pHi-gating. The strength of our approach is that the FRET measurements were performed with simultaneous electrophysiological recordings in inside-out patches with complete control of the intracellular environment. The results showed that the N- and C-termini of the Kir1.1 channel are located closely to each other in the closed (pHi 7.4) state, and move apart when the channel is opened by a high pHi.
Fusion of channels with fluorescent proteins
cDNAs for Kir 1.1 and Kir2.1 channels and the mutant fused with ECFP/EYFP were constructed using commercially available pCMV-ECFP/EYFP vectors (Clontech, Palo Alto, CA). A GGGGGG linker was used to fuse the EYFP to the C terminus of the Kir1.1 and Kir2.1 channels.
HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Sigma Chemical, St. Lois, MO, USA) containing 10% fetal bovine serum (Life Technologies, Paisley, Scotland) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were plated on poly L-lysine-coated No. 1 glass cover slips (42 mm) (Carl Zeiss, Inc., German) and transiently transfected with 2 μg of plasmids using LipofectAMINE 2000 (Invitrogen Co., Carlsbad, CA, USA) and were used 1–2 days after transfection.
Preparation of Xenopus oocytes
Xenopus oocytes were isolated by partial ovariectomy from frogs anaesthetized with 0.1% tricaine (3-aminobenzoic acid ethyl ester). The fused channel cDNAs were subcloned into the pGEMHE expression vector and cRNAs were obtained by in vitro transcription (mMessage mMachine, Ambion, Dallas, USA). Oocytes were used 1–3 days after cRNA or cDNA injection.
Preparation of HEK293T cell isolated plasma membrane sheets
Isolated plasma membrane sheets, attached to the cover slip, were prepared by sonication in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) at 0°C, and incubated at room temperature for 10 minutes before experiments.
Macroscopic currents were recorded at room temperature (21–24°C) using the patch-clamp technique [8, 9] and an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). The internal (pH 6 – 10) and external (pH 7.4) 100 mM [K+] solution contained 80 mM (KCl + KOH), 5 mM EDTA, and 5 mM HEPES. The command voltage pulses were controlled and data acquired using pClamp6 software (Axon Instruments, Foster City, CA, USA).
In Equation  and the definitions of RD1, RD2, and RA1, given below, SFRET, SECFP, and SEYFP followed by D, A, or DA in parenthesis indicate the fluorescence intensity using the indicated filter cube and cells expressing only the donor, ECFP (D), only the acceptor, EYFP (A), or both (DA). RD1, which is equal to SFRET(D)/SECFP(D), RD2, which is equal to SEYFP(D)/SECFP(D), and RA1, which is equal to SFRET(A)/SEYFP(A), are predetermined constants from measurements applied to single cells expressing only ECFP- or EYFP-tagged Kir2.1. RD1, RD2, and RA1 were determined to be 0.51 ± 0.01 (n = 9), 0.0053 ± 0.0001 (n = 9), and 0.26 ± 0.001 (n = 6), respectively. The effective FRET efficiency (EEFF) is determined by
EEFF = (FR - 1)[εYFP(440)/εCFP(440)] (2)
A decrease in the pHi results in a decrease in ECFP and EYFP fluorescence intensity
Effects on the pHi-gating of fusing ECFP and EYFP to the Kir1.1 channel
The FRET efficiency of the ECFP-S219R-EYFP construct is reduced when the channel is in the open state
Based on FRET measurement, it has been suggested that the opening of the Kir3.x channel upon G-protein stimulation induces a rotation of the C-terminal and a decrease in the distance between the N- and C-termini of adjacent subunits . In contrast, we showed that the distance between the N- and C-termini increased when the Kir1.1 channel was open (high pHi). These results support the hypothesis that ligands gating Kir channels initiate conformational changes in the cytoplasmic domains that are transduced to the transmembrane helices and thereby open or close the pore . However, the details of the conformational changes in the cytoplasmic pore in response to ligand gating may vary in different Kir channels. Further experiments are required to understand how the movements of the cytoplasmic pore are transduced to the transmembrane helices.
We thank Drs. Lily Jan and James Weiss for kindly providing the Kir2.1 and Kir1.1 clone, respectively. This work was supported by the Academia Sinica and by the National Science Council of Taiwan (grant 95-2320-B-001-028-MY3).
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