Crystal structure and mechanism of a calcium-gated potassium channel Youxing Jiang, Alice Lee, Jiayun Chen, Martine Cadene, Brian T. Chait & Roderick MacKinnon Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics and Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, 1230 York Avenue, New York, New York 10021, USA ........................................................................................................................................................................................................................... Ion channels exhibit two essential biophysical properties that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus: ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K1 channel that opens in response to intracellular Ca21. We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K1 channel (MthK) from Methanobacterium thermo- autotrophicum in the Ca21-bound, opened state. Eight RCK domains (regulators of K1 conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca21 binding in a simple manner to perform mechanical work to open the pore. Ion channels are central to a wide range of biological processes including cell volume regulation, movement and electrical signal generation1. Ion channel proteins span the membrane of a cell, forming a conduction pathway, or pore, through which ions diffuse down their electrochemical gradient across the membrane. To understand how an ion channel operates as a molecular machine we addressed two mechanistic issues. First, how do ions flow selectively through the pore, and second, how does the pore gate, or open, in response to the appropriate stimulus? For K�� ion channels, significant progress has been made towards understand- ing the mechanism of selective ion conduction2���4. Here we address the mechanism of opening in a ligand-gated K�� channel. K�� channels belong to a family of ion channels called tetrameric cation channels. The family includes K��, Na��, Ca2��, cyclic nucleo- tide-gated, and several other ion channels. They contain four membrane-spanning subunits or domains surrounding a central pore that is selective for cations of one kind or another. On the basis of the KcsA K�� channel structure2,4, it seems that cation selectivity is an intrinsic property of the pore architecture, which provides a special arrangement of cation-attractive ���pore��� a-helices probably shared by all tetrameric cation channel family members. Gating in the tetrameric cation channels is conferred through the attachment of gating domains to the pore. In channels whose gate opens in response to the membrane voltage (voltage-dependent channels), an integral membrane ���voltage sensor��� domain is present on each subunit5,6. In ligand-gated channels, ligand-binding domains are attached to the pore in the aqueous solution near the membrane surface7���11 (Fig. 1). The basic function of these gating domains is to perform mechanical work on the ion conduction pore to change its conformation between closed and opened states. Thus, a voltage sensor converts energy stored in the membrane electric field into mechanical work, whereas ligand-binding domains con- vert the free energy of ligand binding into mechanical work. Thus, ion channel gating comes down to electromechanical or chemo- mechanical coupling between a gating unit and the pore unit. Regulators of K�� conductance (RCK) domains are found in many ligand-gated K�� channels, most often attached to the intracellular carboxy terminus (Fig. 2a)11. These domains are prevalent among prokaryotic K�� channels, and are also found in eukaryotic, high- conductance Ca2��-activated K�� channels (BK channels). Several RCK domain amino-acid sequences are shown in Fig. 2a. The X-ray structure of one RCK domain, that from an Escherichia coli K�� channel, reveals ana-b-protein with a fold similar to dehydrogenase enzymes11. The domain forms a homodimer, producing a cleft between two lobes. The dimer is similar in its structure to certain bi- lobed, amino acid and nutrient molecule-binding proteins found in the periplasm of bacteria12���14. We postulated that the E. coli K�� channel binds a ligand molecule in the cleft to affect channel gating. On the basis of amino-acid sequence analysis it is apparent that certain RCK domains bind nicotinamide adenine dinucleotide (NAD)15,16, but for many RCK domains the ligand is unknown. Analysis of DNA sequences shows that RCK domains occur in at least five different contexts (Fig. 2b): (1) as a single domain on the C terminus of a K�� channel (for example, many prokaryotic K�� channels) (2) as two tandem RCK domains on the C terminus (for example, eukaryotic BK channels) (3) as two domains, one on the N and another on the C terminus (4) as a soluble protein (not part of a K�� channel gene) consisting of two tandem RCK domains and (5) as a soluble protein consisting of a single RCK domain. Of note, in three out of five contexts (Fig. 2b, (2)���(4)), RCK domains occur in pairs. The MthK channel We cloned and expressed a K�� channel gene from the archeon Methanobacterium thermoautotrophicum. The gene codes for a K�� channel that we have named MthK it contains two membrane- spanning segments per subunit, which forms one subunit of the transmembrane pore, and a single C-terminal RCK domain (Fig. 3a). Expression in E. coli, extraction with decylmaltoside Figure 1 Ligand-activated ion channel gating. A ligand (black oval) binds to receptor domains, inducing a conformational change that leads to opening of the ion conduction pore. The ligand receptor is usually located at the membrane surface on the extracellular side (top) in neurotransmitter-gated ion channels and on the intracellular side (bottom) in second-messenger-gated ion channels, as shown here. articles NATURE | VOL 417 | 30 MAY 2002 | www.nature.com 515 �� 2002 Nature Publishing Group

(DM), and purification yields a protein���detergent complex that elutes in a peak at approximately 11 ml on a superdex-200 (10/30) gel filtration column (Fig. 3b). Protein from this peak runs in two bands near a relative molecular mass of 26,000 and 200,000 (M r 26K and 200K, respectively) on SDS���polyacrylamide gel electrophoresis (PAGE) (Fig. 3b). Mass spectrometry shows that the approximately 200K band contains the full-length gene product, presumably migrating as a tetramer on SDS���PAGE, and that the roughly 26K band contains only the C-terminal RCK domain, beginning at amino-acid position Met 107. In separate experiments we observed a similar result on overexpression of the E. coli K�� channel in E. coli: two proteins were produced, one full length and another corre- sponding to a C-terminal RCK domain, beginning at an internal methionine residue. We reasoned that a single gene gives rise to two gene products, and that both products are required in the assembly of a functional K�� channel (Fig. 3d). Mutation of the hypothesized internal start site, Met 107, to Ile (M107I) eliminated the 26K gene product (Fig. 3c) and resulted in reduced levels of MthK channel expression. Curiously, on the superdex-200 column the mutant channel elutes at approximately 10 ml, indicating that it is larger in size than the wild-type channel (Fig. 3c). The structural analysis below offers an explanation for this unexpected result that two mutant channels bind to each other once they have been extracted from the membrane (Fig. 3e). Such binding would provide com- pensation for the missing soluble domains and explain the large protein���detergent complex on gel filtration. Electrophysiological analysis MthK channels produced with Met 107 intact were reconstituted into planar lipid bilayers of 1-palmitoyl-2-oleoyl phosphatidylgly- cerol (POPG) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) (Fig. 4). The channel is selective for K�� over Na�� (data not shown), and in solutions containing 150 mM KCl and 10 mM HEPES, pH 7.0 on both sides of the membrane it exhibits inward rectification: the current���voltage curve is nonlinear, with a greater slope at negative voltages (Fig. 4a). Single channels show a con- ductance of approximately 200 pS at 2100 mV. The K�� channel blocker charybdotoxin (CTX), a protein from scorpion venom17, inhibits the MthK channel, underscoring the structural similarity between this prokaryotic K�� channel and its eukaryotic family members (Fig. 4b). CTX binds to the extracellular pore entryway of K�� channels18, which allows easy assessment of a channel���s orien- tation within the membrane, that is, it distinguishes the extracellu- lar (toxin sensitive) and intracellular (toxin insensitive) sides. Figure 4b shows an important property of the MthK channel. When Ca2�� is applied to the intracellular solution, the probability of channel opening increases (Fig. 4b). The ���0 mM��� Ca2�� recording contains trace amounts of Ca2�� necessary to open channels in the presence of EDTA, a Ca2�� chelator, MthK channels essentially never open. Thus, Ca2�� regulates or gates the channel from the intracellular solution: the effect of Ca2�� continues into the milli- molar concentration range. The reduced single-channel amplitude at higher Ca2�� concentrations is due to rapid pore block by Ca2�� ions, a well known phenomenon19���21. The principal result, however, is that Ca2�� gates the channel open. We do not know whether Ca2�� is a physiological ligand for gating the MthK channel, but for the purposes of the present study, Ca2�� is a ligand that opens the channel in a concentration-dependent manner. X-ray structure analysis We solved the three-dimensional structure of the MthK channel by X-ray crystallography at a resolution of 3.3 A �� (Methods see also Figure 2 Sequence analysis of proteins containing RCK domains. a, Partial sequence alignment of K�� channels and prokaryotic K�� transporters (TrkA). The alignment begins at the K�� channel signature sequence (Filter) and ends at the MthK channel (MthK2TM) C terminus. Secondary structure assignment is based on the MthK crystal structure: blue bars and arrows showa-helices andb-strands, respectively dashed line shows the structurally undefined linker connecting the pore to the RCK domain. Cyan indicates semi- conserved sequence red shows an NAD-binding motif present in some RCK domains. K�� channels: MthK2TM, M. thermautotrophicum (GI:2622639) A.aeo2TM, Aquifex aeolicus (GI:2983007) M.jan2TM, Methanococcus jannaschii (GI:1498918) S.sp2TM, S. sp (GI:7447543) E.coli6TM, E. coli (GI 400124) Y.pest6TM, Yersinia pestis (sanger_632) TrkA proteins: E.coliTrkA2, E. coli, domain 2 (GI:136235) P.abyTrkA, Pyrococcus abyssi (GI:7450648) BK K�� channels: HumanBK, Homo sapiens (GI:2570854) FlyBK, Drosophila melanogaster (GI:7301192). b, RCK domains: (1) single C-terminal domain of K�� channels (2) tandem C-terminal domains (BK channels) (3) N- and C-terminal domains (Synechocystis sp. channel) and (4) and (5) soluble containing tandem (E. coli TrkA) or single (P. abyssi TrkA) domains of prokaryotic K�� transporters. articles NATURE | VOL 417 | 30 MAY 2002 | www.nature.com 516 �� 2002 Nature Publishing Group