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• W2113456482 abstract "Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K+ channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP2 and Mg2+-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels. Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K+ channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP2 and Mg2+-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels. GTP-binding (G) 6The abbreviations used are: GGTP-bindingPIP2phosphatidylinositol 4,5-bisphosphateDDMdodecyl maltosideGTPγSguanosine 5′-3-O-(thio)triphosphateEMelectron microscopyPLproteoliposomesPEphosphatidylethanolaminePSphosphatidylserineFSCFourier shell correlation. protein-sensitive potassium (K+) channels comprise the third subfamily of inwardly rectifying (Kir) channels, so called as they conduct more current in the inward than outward direction. Like all Kir family members, Kir3 channels depend on phosphoinositides to maintain their activity (1Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 2Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1307-1312Crossref PubMed Scopus (213) Google Scholar, 3Logothetis D.E. Lupyan D. Rosenhouse-Dantsker A. J. Physiol. 2007; 582: 953-965Crossref PubMed Scopus (46) Google Scholar). Kir3 channels are unique among other Kir members in that their activity is stimulated by the βγ subunits of G proteins (Gβγ) (4Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (877) Google Scholar, 5Reuveny E. Slesinger P.A. Inglese J. Morales J.M. Iñiguez-Lluhi J.A. Lefkowitz R.J. Bourne H.R. Jan Y.N. Jan L.Y. Nature. 1994; 370: 143-146Crossref PubMed Scopus (422) Google Scholar). Indeed, a wide variety of G protein-coupled receptors activate Kir3 channels, including the M2-muscarinic, opioid, 5-HT serotonin, A1-adenosine, α2-adrenergic, D2-dopamine, and GABAB receptors (6Yamada M. Innabe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-760PubMed Google Scholar). Kir3 channels play an important role in human physiology as they can control heart rate and neuronal excitability (7Hibino H. Inanobe A. Furutani K. Murakami S. Findlay I. Kurachi Y. Physiol. Rev. 2010; 90: 291-366Crossref PubMed Scopus (1054) Google Scholar). GTP-binding phosphatidylinositol 4,5-bisphosphate dodecyl maltoside guanosine 5′-3-O-(thio)triphosphate electron microscopy proteoliposomes phosphatidylethanolamine phosphatidylserine Fourier shell correlation. A number of comprehensive Kir reviews summarize numerous background studies on this type of K+ channels (e.g. Refs. 7Hibino H. Inanobe A. Furutani K. Murakami S. Findlay I. Kurachi Y. Physiol. Rev. 2010; 90: 291-366Crossref PubMed Scopus (1054) Google Scholar, 8Stanfield P.R. Nakajima S. Nakajima Y. Rev. Physiol. Biochem. Pharmacol. 2002; 145: 47-179Crossref PubMed Google Scholar). Four mammalian Kir3 members have been identified (Kir3.1–3.4) (9Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (546) Google Scholar, 10Dascal N. Lim N.F. Schreibmayer W. Wang W. Davidson N. Lester H.A. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 6596-6600Crossref PubMed Scopus (81) Google Scholar, 11Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar, 12Krapivinsky G. Gordon E.A. Wickman K. Velimirović B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (758) Google Scholar). Kir channels consist of a pore (P) region flanked by two transmembrane domains (M1 and M2). A recent crystallographic structure of Kir2.2 (13Tao X. Avalos J.L. Chen J. MacKinnon R. Science. 2009; 326: 1668-1674Crossref PubMed Scopus (275) Google Scholar) confirmed a similar architecture for the transmembrane portion of a mammalian Kir channel compared with bacterial channels, such as the KcsA, KirBac1.1, and KirBac3.1 (14Doyle D.A. Cabral J.M. Pfeutzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. Mackinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5747) Google Scholar, 15Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; 300: 1922-1926Crossref PubMed Scopus (738) Google Scholar, 16Kuo A. Domene C. Johnson L.N. Doyle D.A. Vénien-Bryan C. Structure. 2005; 13: 1463-1472Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). High resolution structures of a chimera (hereafter referred to as the Kir3.1 chimera) between the cytosolic region of Kir3.1 and the transmembrane region of a prokaryotic Kir channel (KirBac1.3) have indeed captured one of the putative cytosolic gates (the G-loop gate) in two states, seemingly “open” and “closed” (17Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (258) Google Scholar). Structures of complexes of Kir3 channel intracellular domains (18Nishida M. MacKinnon R. Cell. 2002; 111: 957-965Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 19Pegan S. Arrabit C. Zhou W. Kwiatkowski W. Collins A. Slesinger P.A. Choe S. Nat. Neurosci. 2005; 8: 279-287Crossref PubMed Scopus (255) Google Scholar, 20Inanobe A. Matsuura T. Nakagawa A. Kurachi Y. Channels. 2007; 1: 39-45Crossref PubMed Scopus (38) Google Scholar) or the Kir3.1 chimera with the Gβγ subunits have not yet been elucidated, presumably because of their low stability. Kir3.1 channels do not form functional homomers and they localize poorly to the cell surface (e.g. Ref. 21Mirshahi T. Logothetis D.E. J. Biol. Chem. 2004; 279: 11890-11897Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Yet, they potentiate the activity of other Kir3 channels upon assembly into heteromeric complexes (e.g. Refs.12Krapivinsky G. Gordon E.A. Wickman K. Velimirović B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (758) Google Scholar, 22Lesage F. Guillemare E. Fink M. Duprat F. Heurteaux C. Fosset M. Romey G. Barhanin J. Lazdunski M. J. Biol. Chem. 1995; 270: 28660-28667Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Kir3 channels, other than Kir3.1, also exist as homotetramers (e.g. Kir3.2 or Kir3.4) (11Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar, 23Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14193-14198Crossref PubMed Scopus (99) Google Scholar), albeit exhibiting lower activity than when found in heteromeric complexes with Kir3.1. Specific point mutations in a pore helix position of Kir3.1 (F137S or Kir3.1*) and Kir3.4 (S143T) have yielded potentiated homomeric currents with qualitatively similar properties to the wild-type heteromeric Kir3.1/3.4 currents (23Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14193-14198Crossref PubMed Scopus (99) Google Scholar, 24Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Kir3 channels are highly expressed in heart (Kir3.1, Kir3.4) and brain (Kir3.1, Kir3.2, Kir3.3). Phosphoinositides regulate the activity of many different ion channels and transporters (e.g. 25–26). Phosphoinositide dependence of Kir channels has been studied extensively (25Logothetis D.E. Petrou V.I. Adney S.K. Mahajan R. Pflugers Arch. 2010; 460: 321-341Crossref PubMed Scopus (89) Google Scholar, 26Logothetis D.E. Nilius B. Pflügers Arch. 2007; 455: 1-3Crossref PubMed Scopus (15) Google Scholar, 27Logothetis D.E. Jin T. Lupyan D. Rosenhouse-Dantsker A. Pflügers Arch. 2007; 455: 83-95Crossref PubMed Scopus (107) Google Scholar). A model emerging from such studies proposes that opening of the cytosolic gates occurs as the cytosolic domains of Kir channels get tethered to the plasma membrane by virtue of electrostatic interactions between the acidic phosphoinositides and basic binding pockets on the channel surface near the inner leaflet of the lipid bilayer (26Logothetis D.E. Nilius B. Pflügers Arch. 2007; 455: 1-3Crossref PubMed Scopus (15) Google Scholar, 28Stansfeld P.J. Hopkinson R. Ashcroft F.M. Sansom M.S. Biochemistry. 2009; 48: 10926-10933Crossref PubMed Scopus (109) Google Scholar). High affinity of channel-PIP2 interactions correlates strongly with high channel activity (29Lopes C.M. Zhang H. Rohacs T. Jin T. Yang J. Logothetis D.E. Neuron. 2002; 34: 933-944Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). It has been suggested that channel-PIP2 interactions affect the cytosolic G-loop gate (19Pegan S. Arrabit C. Zhou W. Kwiatkowski W. Collins A. Slesinger P.A. Choe S. Nat. Neurosci. 2005; 8: 279-287Crossref PubMed Scopus (255) Google Scholar) and that mutations that cause disease alter channel-PIP2 interactions (25Logothetis D.E. Petrou V.I. Adney S.K. Mahajan R. Pflugers Arch. 2010; 460: 321-341Crossref PubMed Scopus (89) Google Scholar, 29Lopes C.M. Zhang H. Rohacs T. Jin T. Yang J. Logothetis D.E. Neuron. 2002; 34: 933-944Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Furthermore ethanol has been shown to activate Kir3 channels (6Yamada M. Innabe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-760PubMed Google Scholar, 7Hibino H. Inanobe A. Furutani K. Murakami S. Findlay I. Kurachi Y. Physiol. Rev. 2010; 90: 291-366Crossref PubMed Scopus (1054) Google Scholar). The structure of the Kir3.1 chimera (17Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (258) Google Scholar) is the first high resolution Kir3 structure that contains both cytosolic and transmembrane channel domains. The chimera contains the Kir3.1 residues Lys41–Trp82 (N terminus) and Phe181–Leu371 (bottom of M2 and C terminus) and the KirBac1.3 residues Phe45–Ala127 (transmembrane domains and extracellular loops). Thus, the chimera is missing the transmembrane domains (Asn83–Met180) and the last 129 C-terminal residues (Ile372–Thr501) of Kir3.1. The lack of functional expression or reconstitution of activity of this chimeric channel casted doubt as to its usefulness in being utilized as a model for Kir3 structure and function studies. Here, we aimed to functionally reconstitute the purified Kir3.1 chimera in planar lipid bilayers and to test its sensitivity to molecules that modulate Kir3 activity, such as phosphoinositides, ethanol and G proteins. A three-dimensional reconstruction of the Kir3.1 chimera by single particle electron microscopy was consistent with the crystal structure. Purified Kir3.1 chimera displayed activity only in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2). Ethanol stimulated the activity of the Kir3.1 chimera, consistent with its effect on wild-type Kir3 currents. Interestingly, the activity of the Kir3.1 chimera was inhibited rather than stimulated by nanomolar concentrations of Gβγ or Gα-GDP or Gα-GTPγS. Yet, activity of the Kir3.1 chimera was stimulated in the presence of both activated G-protein subunits (i.e. Gα-GTPγS and Gβγ). Such stimulation recovered approximately half of the PIP2-induced activity that had been inhibited by the individual G protein subunits or the heterotrimeric complex. These results pave the way for future electrophysiology and structural studies of the Kir3.1 chimera in complex with the G protein subunits aimed at understanding the molecular basis of Kir3 channel regulation. The expression and purification of the Kir3.1 chimera was carried out following a previously described protocol (17Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (258) Google Scholar) with the following modifications: 1) Following incubation with thrombin to remove the His tag the sample was run over a high-affinity cobalt resin (Clontech) for the second time to eliminate uncleaved material and impurities. 2) Size-exclusion chromatography was carried out using a Sephacryl S-200 gel filtration column equilibrated with buffer (8 mm Bis-Tris, pH 6.5, 120 mm KCl, 3 mm DTT, and 5 mm DDM) containing the detergent dodecyl maltoside (DDM). The identity of the Kir3.1 chimera was confirmed by in-gel digestion and mass spectrometry. The yield was ∼0.5 mg of pure Kir3.1 chimera per liter of Escherichia coli culture. The Kir3.1 chimera that eluted in peak B (supplemental Fig. S1A) was diluted (1/20) in gel filtration buffer. A 2-μl aliquot of the dilution was adsorbed onto glow-discharged carbon-coated copper grids, and negatively stained with 2% uranyl acetate. The specimen was imaged in a Jeol 2100F FEG transmission electron microscope at 200 kV under low dose conditions, using a 2k × 2k pixel CCD camera at the equivalent calibrated magnification of 63,450. To avoid biases generated during manual particle selection, we employed a strategy that involves: automated particle selection (30Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2102) Google Scholar), followed by statistical analysis, alignment and classification (31Marabini R. Masegosa I.M. San Martin M.C. Marco S. Fernandez J.J. de, la, Fraga L.G. Vaquerizo C. Carazo J.M. J. Struct. Biol. 1996; 116: 237-240Crossref PubMed Scopus (171) Google Scholar, 32Scheres S.H. Núñez-Ramírez R. Sorzano C.O. Carazo J.M. Marabini R. Nat. Protoc. 2008; 3: 977-990Crossref PubMed Scopus (275) Google Scholar). An initial dataset of 51,000 particles was automatically selected from 130 CCD images using EMAN (30Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2102) Google Scholar). The software Xmipp (31Marabini R. Masegosa I.M. San Martin M.C. Marco S. Fernandez J.J. de, la, Fraga L.G. Vaquerizo C. Carazo J.M. J. Struct. Biol. 1996; 116: 237-240Crossref PubMed Scopus (171) Google Scholar, 32Scheres S.H. Núñez-Ramírez R. Sorzano C.O. Carazo J.M. Marabini R. Nat. Protoc. 2008; 3: 977-990Crossref PubMed Scopus (275) Google Scholar) was employed to extract particles in 64 × 64 images, to normalize them and to perform statistical analysis. Following normalization ∼5% of the initial images were discarded using purely statistical criteria based on the standard deviation of the dataset. The contrast transfer function of the images was estimated using CTFFIND3 (33Mindell J.A. Grigorieff N. J. Struct. Biol. 2003; 142: 334-347Crossref PubMed Scopus (1142) Google Scholar) and corrected using Bsoft (34Heymann J.B. J. Struct. Biol. 2001; 133: 156-169Crossref PubMed Scopus (195) Google Scholar). Subsequently, the particles were grouped into 24 different defocus groups to perform classification and heterogeneity analysis. Alignment of images, two-dimensional and three-dimensional maximum likelihood classification, and reconstruction were performed using Xmipp. To obtain representative families of the heterogeneity present in the specimen, we carried out five successive rounds of MLF2D (multireference two-dimensional alignment using maximum-likelihood in Fourier space), a maximum likelihood algorithm included in the Xmipp package (31Marabini R. Masegosa I.M. San Martin M.C. Marco S. Fernandez J.J. de, la, Fraga L.G. Vaquerizo C. Carazo J.M. J. Struct. Biol. 1996; 116: 237-240Crossref PubMed Scopus (171) Google Scholar, 32Scheres S.H. Núñez-Ramírez R. Sorzano C.O. Carazo J.M. Marabini R. Nat. Protoc. 2008; 3: 977-990Crossref PubMed Scopus (275) Google Scholar). During this process, particle images with an irregular background, close neighbors, overlapping particles and aggregates were discarded to yield a homogeneous dataset of 19,300 particles. Heterogeneity analysis and three-dimensional reconstruction was carried out using MLF3D (35Scheres S.H. Núñez-Ramírez R. Gómez-Llorente Y. San Martín C. Eggermont P.P. Carazo J.M. Structure. 2007; 15: 1167-1177Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To this end, an initial volume was generated by the common lines method using EMAN and without imposing any symmetry. This volume was then filtered to a resolution of 80 Å, and its gray scale corrected according to the protocol recommended by the developers of MLF3D. From this model, 3 initial seeds were generated (using different subsets from the 19,300 particle dataset) to serve as initial volumes for MLF3D. Following 25 iterations, in which no symmetry was imposed, the two most populated volumes (containing 52 and 34% of the particles) were used to generate 4 new initial seeds for a new round of MLF3D. The resulting four volumes were very similar and their back-projections were comparable to reference-free average classes solved by MLF2D. The first volume, containing 35% of the particle images (6,900 out of 19,300) and an estimated resolution of 24 Å, was selected to calculate the final reconstruction by imposing 4-fold symmetry around the z axis. The 0.5 criterion of the Fourier shell correlation (FSC) was employed to estimate the resolution of the final map. Bilayer experiments were performed as described (36Leal-Pinto E. London R.D. Knorr B.A. Abramson R.G. J. Membr. Biol. 1995; 146: 123-132Crossref PubMed Scopus (13) Google Scholar, 37Leal-Pinto E. Tao W. Rappaport J. Richardson M. Knorr B.A. Abramson R.G. J. Biol. Chem. 1997; 272: 617-625Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly, purified Kir3.1 chimera was used to form proteoliposomes (PLs) by sonicating the purified protein with a 1:1 mixture of bovine brain phosphatidylethanolamine (PE: 10 mg/ml) and phosphatidylserine (PS: 10 mg/ml). The experimental apparatus consisted of two 1-ml buffer chambers separated by a Teflon film that contained a single 50–100-μm hole. A lipid bilayer was formed by “painting” the hole with a 1:1 mixture of PE and PS dissolved in n-decane to a final concentration of 50 μg/ml. This resulted in formation of a high-resistance seal between the two cups. For these studies, the Cis side was defined as the chamber connected to the voltage-holding electrode, and all voltages are referenced to the Trans (ground) chamber. Stability of the bilayer was determined by clamping the voltage at various levels. If a resistance was >100 GΩ, the noise <0.2 pA, and the bilayer was stable 5 μl aliquots of PLs containing the Kir3.1 chimera were added to the Trans side of the chamber and stirred for 5 min. When channel activity was observed, PLs were washed from the Trans chamber to limit further channel incorporation. The orientation of the Kir3.1 chimera insertion was with the intracellular surface facing the Cis side of the bilayer. We attribute our high success of channel insertion at the appropriate orientation to the asymmetry of the lipids used (38Rostovtseva T.K. Kazemi N. Weinrich M. Bezrukov S.M. J. Biol. Chem. 2006; 281: 37496-37506Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Records were filtered at 10 kHz, unless otherwise indicated. Channel events with an open time greater than 2.0 ms and a noise level at the open state less than two times the background noise were further filtered at 1 kHz for further analysis (see below). All experiments were performed with either symmetrical buffered solutions or with ionic gradients to examine channel selectivity. Exact composition of solutions used in each experiment is described below. For all of the experiments (Kir3.1 chimera or oocytes membranes) the solutions were similar containing in mm: 150 KCl, 1 CaCl2, 1 MgCl2, 1% Chaps, 10 Tris-Hepes, pH 7.4. The solutions were symmetrical in both sides of the chamber unless otherwise indicated. The bilayer channel events were analyzed using the Clampfit module (version 9.2.1.9) of pClamp (Axon Inc.). The software determines the valid channel transitions (i.e. openings and closings), based on 50% threshold crossing methods. If multiple channel events are observed in a single patch/recording, the total number of functional channels (N) in the patch can be estimated from the number of peaks in the all point amplitude histogram. In such cases, the product of number of channels (N) and the open probability (Po) can be used to measure the channel activity in the patch. In the records shown in Fig. 5, C–F, NPo was obtained in 10 s sequential intervals throughout the experiment. For each condition, the reagent indicated was added to either the Cis or Trans side of the bilayer as indicated and the resulting NPo was normalized to the corresponding NPo for PIP2. Percent NPo data were pooled together in the summary graphs shown in 5D, F, which plot mean ± S.E. values. Statistical significance shown for these plots was obtained using one-way ANOVA analysis in MicroCal Origin 7.5. The Kir3.1 chimera was expressed in E. coli and purified (supplemental Fig. S1) in DDM, following a protocol described by Nishida et al. (17Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (258) Google Scholar). To ensure that the purified protein to be employed in our electrophysiology experiments retained its tetrameric assembly and structural integrity, we performed a three-dimensional reconstruction of the chimera using single particle electron crystallography. Fig. 1A displays a representative field view showing abundant globular particles with a diameter of ∼100 Å (see “Experimental Procedures”). The remaining subset of particle images (19,300 particles, ∼38% of the initial dataset) displayed a very high correlation in terms of size and background. The reference-free class averages generated for this subset appeared as different projections from the same object (Fig. 1B), and displayed an enrichment of lateral orientations (supplemental Fig. S2A). This subset of 19,300 particle images was employed for three-dimensional reconstruction and refinement, coupled with heterogeneity analysis (35Scheres S.H. Núñez-Ramírez R. Gómez-Llorente Y. San Martín C. Eggermont P.P. Carazo J.M. Structure. 2007; 15: 1167-1177Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Fig. 1C illustrates the reconstructed volume after applying a 4-fold symmetry parallel to the z axis. Back-projections of the three-dimensional reconstruction correspond closely with reference-free class averages (supplemental Fig. S2B), indicating consistency between the reconstructed structure and the particle dataset. The final structure (Fig. 1C) was determined to a resolution of 24 Å based on the 0.5 criterion of the FSC (supplemental Fig. S2C). Fig. 1D displays a cut-away view to show the fitting of the crystal structure of the Kir3.1 chimera tetramer (PDB code: 2QKS) solved by Nishida et al. (17Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (258) Google Scholar). Overall, our EM map was consistent with the x-ray structure of the tetrameric chimera, indicating that the protein employed in our experiments was indeed tetrameric. Notably, the cytoplasmic region of the x-ray structure fitted very well (manual fitting using CHIMERA (39Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (28368) Google Scholar)) within the envelope of our reconstruction. In the transmembrane region a semispherical additional mass could be observed likely corresponding to DDM molecules arranged concentrically around the transmembrane helices of the chimera. We note that comparable features due to bound detergent have been observed in single particle EM structures of detergent-solubilized membrane proteins both under negative stain (40Rubinstein J.L. Methods. 2007; 41: 409-416Crossref PubMed Scopus (32) Google Scholar) and in vitreous ice (41Muench S.P. Huss M. Song C.F. Phillips C. Wieczorek H. Trinick J. Harrison M.A. J. Mol. Biol. 2009; 386: 989-999Crossref PubMed Scopus (82) Google Scholar). Nishida et al. (17Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (258) Google Scholar) concluded their structural study of the Kir3.1 chimera unable to obtain functional reconstitution of this protein. They had attempted functional reconstitution in planar lipid membranes consisting of POPE:POPG lipids in a 3:1 ratio and speculated several potential reasons for the lack of function. 1) There could have been an unmet lipid requirement. 2) The chimera could have been non-functional as a homomultimer, because Kir3.1 is normally functional as a heteromultimer with other members of the Kir3 family. 3) The chimera might have lacked the proper coupling between the cytoplasmic and transmembrane pores; and 4) the prokaryotic transmembrane domain of the chimera could have been problematic for functional reconstitution into the bilayer system, as single channel activity had not been demonstrated for any of the prokaryotic Kir channels. Because Kir3.1 is found as a complex with Kir3.4 in atrial cells giving rise to KAch, we first set out to test whether the Kir3.1 chimera could be functionally expressed in Xenopus laevis oocytes or HEK-293 cells, either by itself or in complex with Kir3.4 subunits. Injection into Xenopus oocytes of the Kir3.1 chimera mRNA alone (supplemental Fig. S3, B and F) or together with Kir3.4 mRNA (supplemental Fig. S2, D and F) yielded no significantly increased currents compared with the muscarinic type 2 receptor (M2R control) injected alone (supplemental Fig. S3, A and F) or together with twice the amount of Kir3.4 (Kir3.4 control) (supplemental Fig. S3, C and F). In contrast, co-injection of wild-type Kir3.1 and Kir3.4 mRNAs resulted in significantly higher currents than any of the homomeric subunit injections alone, consistent with previous results (supplemental Fig. S3, E and F) (12Krapivinsky G. Gordon E.A. Wickman K. Velimirović B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (758) Google Scholar, 42Chan K.W. Langan M.N. Sui J.L. Kozak J.A. Pabon A. Ladias J.A. Logothetis D.E. J. Gen. Physiol. 1996; 107: 381-397Crossref PubMed Scopus (67) Google Scholar). Tagging of the C-terminal cytoplasmic tail of the Kir3.1 chimera with EGFP (Kir3.1 Chim-GFP) and transfecting HEK-293 cells, revealed lack of cell surface expression (supplemental Fig. S4A), similar to that previously observed with Kir3.1-GFP alone (e.g. 24). Even co-transfection of Kir3.4 failed to alter cell surface expression of the Kir3.1 Chim-GFP (supplemental Fig. S4B), in sharp contrast with Kir3.1-GFP shown previously (21Mirshahi T. Logothetis D.E. J. Biol. Chem. 2004; 279: 11890-11897Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). These results are consistent with the interpretation that the Kir3.1 chimera neither produces functional homomeric channels nor it localizes to the cell surface. In addition, the chimera failed to show potentiated currents when expressed together with Kir3.4 and to be localized to the cell surface, suggesting a possible failure to associate with Kir3.4. Indeed, the Kir3.1 chimera is missing the 40 amino acid residues of N-terminal end of Kir3.1 that have been shown previously to be critical for heteromeric assembly with Kir3.4 and cell surface localization (21Mirshahi T. Logothetis D.E. J. Biol. Chem. 2004; 279: 11890-11897Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Following these unsuccessful attempts to attain functional expression from the" @default.
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