M2 Helix Research Articles

Amino Acid Substitutions in the Pore Helix of GluR6 Control Inhibition by Membrane Fatty Acids

RNA editing at the Q/R site in the GluR5 and GluR6 subunits of neuronal kainate receptors regulates channel inhibition by lipid-derived modulators including the cis-unsaturated fatty acids arachidonic acid and docosahexaenoic acid. Kainate receptor channels in which all of the subunits are in the edited (R) form exhibit strong inhibition by these compounds, whereas wild-type receptors that include a glutamine (Q) at the Q/R site in one or more subunits are resistant to inhibition. In the present study, we have performed an arginine scan of residues in the pore loop of the GluR6(Q) subunit. Amino acids within the range from −19 to +7 of the Q/R site of GluR6(Q) were individually mutated to arginine and the mutant cDNAs were expressed as homomeric channels in HEK 293 cells. All but one of the single arginine substitution mutants yielded functional channels. Only weak inhibition, typical of wild-type GluR6(Q) channels, was observed for substitutions +1 to +6 downstream of the Q/R site. However, arginine substitution at several locations upstream of the Q/R site resulted in homomeric channels exhibiting strong inhibition by fatty acids, which is characteristic of homomeric GluR6(R) channels. Based on homology with the pore loop of potassium channels, locations at which R substitution induces susceptibility to fatty acid inhibition face away from the cytoplasm toward the M1 and M3 helices and surrounding lipids.

Theoretical Studies on the Structure and Symmetry of the Transmembrane Region of Glutamatergic GluR5 Receptor

There is numerous experimental and conceptual proof that the extracellular portion of ionotropic glutamate receptors (iGluRs), i.e., N-terminal domain (NTD) and ligand-binding core (LBD), exhibits 2-fold rotational symmetry and thus dimer of dimers architecture. However, the problem of the structure and symmetry of the transmembrane channel forming region of iGluRs has not been solved yet. According to the most common approach, glutamate ion channels possess 4-fold symmetry, similar to homologous potassium channels. This results in a symmetry mismatch between the extracellular fragment of the receptor and its transmembrane domain. To overcome the above discrepancies in iGluR symmetry, homology modeling was applied to propose an alternative model of GluR5 channel transmembrane region. Because of modification of M3 helix structure, as indicated by experimental results, the obtained model is generally characterized by 2-fold rotational symmetry. As a validation of the applied methodology, IEM-1754 was docked to the obtained GluR5 receptor transmembrane fragment model. However, because there are no affinity values available for IEM-1754, the applied methodology was additionally validated by building of NMDA receptor transmembrane region model and its evaluation in the docking of dextrorphan, (+)-MK-801, and IEM-1925. Moreover, the NMDA and GluR5 channel models are consistent with all available experimental data, including the latest single-particle electron microscopy images of iGluRs.

Crystal Structure of D351A and P312A Mutant Forms of the Mammalian Sarcoplasmic Reticulum Ca2+-ATPase Reveals Key Events in Phosphorylation and Ca2+ Release

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF(4)(-) bound (3.5-A resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-A resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca(2+) was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca(2+)-translocating Ca(2)E1P-->E2P transition, which seems to be due to a stabilization of Ca(2)E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro(312) with alanine in P312A.

Mechanics of Channel Gating of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer β-sheet composed of β9 and β10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O ↔ C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.

The structural basis of calcium transport by the calcium pump

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the Cys-Loop and M2–M3 Linker

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a “gate” in the transmembrane domain (TMD). We used Φ-value analysis to probe the relative timing of the gating motions of α-subunit residues located near the ECD–TMD interface. Mutation of four of the seven amino acids in the M2–M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (Keq) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Φ-value for the whole linker was ∼0.64. One interpretation of this result is that the gating motions of the M2–M3 linker are approximately synchronous with those of much of M2 (∼0.64), but occur after those of the transmitter binding site region (∼0.93) and loops 2 and 7 (∼0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed Keq by 2800-, 10-, and 18-fold, respectively, and with an average Φ-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (≤0.51 kcal mol−1). The M2–M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an ∼16-Å border and involve about a dozen residues.

Phospholemman Transmembrane Structure Reveals Potential Interactions with Na+/K+-ATPase

Phospholemman (PLM) is a 72-residue bitopic cardiac transmembrane protein, which acts as a modulator of the Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger and possibly forms taurine channels in nonheart tissue. This work presents a high resolution structural model obtained from a combination of site-specific infrared spectroscopy and experimentally constrained high throughput molecular dynamics (MD) simulations. Altogether, 37 experimental constraints, including nine long range orientational constraints, have been used during MD simulations in an explicit lipid bilayer/water system. The resulting tetrameric alpha-helical bundle has an average helix tilt of 7.3 degrees and a crossing angle close to 0 degrees . It does not reveal a hydrophilic pore, but instead strong interactions between various residues occlude any pore. The helix-helix packing is unusual, with Gly(19) and Gly(20) pointing to the outside of the helical bundle, facilitating potential interaction with other transmembrane proteins, thus providing a structural basis for the modulatory effect of PLM on the Na(+)/K(+)-ATPase. A two-stage model of interaction between PLM and the Na(+)/K(+)-ATPase is discussed involving PLM-ATPase interaction and subsequent formation of an unstable PLM trimer, which readily interacts with surrounding ATPase molecules. Further unconstrained MD simulations identified other packing models of PLM, one of which could potentially undergo a conformational transition to an open pore.

Nanosecond-Timescale Conformational Dynamics of the Human α7 Nicotinic Acetylcholine Receptor

We explore the conformational dynamics of a homology model of the human α7 nicotinic acetylcholine receptor using molecular dynamics simulation and analyses of root mean-square fluctuations, block partitioning of segmental motion, and principal component analysis. The results reveal flexible regions and concerted global motions of the subunits encompassing extracellular and transmembrane domains of the subunits. The most relevant motions comprise a bending, hinged at the β10-M1 region, accompanied by concerted tilting of the M2 helices that widens the intracellular end of the channel. Despite the nanosecond timescale, the observations suggest that tilting of the M2 helices may initiate opening of the pore. The results also reveal direct coupling between a twisting motion of the extracellular domain and dynamic changes of M2. Covariance analysis of interresidue motions shows that this coupling arises through a network of residues within the Cys and M2-M3 loops where Phe 135 is stabilized within a hydrophobic pocket formed by Leu 270 and Ile 271. The resulting concerted motion causes a downward shift of the M2 helices that disrupts a hydrophobic girdle formed by 9′ and 13′ residues.

Progesterone binding to the α1-subunit of the Na/K-ATPase on the cell surface: Insights from computational modeling

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [ 3H]ouabain and [ 3H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112 kDa α1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding α1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar β and hydrophobic α surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg 118, Asp 129 and possibly Glu 122–124 in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys 111 near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding.

Proton paths in the sarcoplasmic reticulum Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) pumps Ca2+ and countertransport protons. Proton pathways in the Ca2+ bound and Ca2+-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca2+ bound state Ca2E1, one of the proposed Ca2+ entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca2+-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca2+ binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca2+ dissociation pathways. We suggest that separate proton and Ca2+ pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.

3H]Benzophenone Photolabeling Identifies State-Dependent Changes in Nicotinic Acetylcholine Receptor Structure

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.

Domain Organization and Movements in Heavy Metal Ion Pumps

To study domain organization and movements in the reaction cycle of heavy metal ion pumps, CopA, a bacterial Cu+-ATPase from Thermotoga maritima was cloned, overexpressed, and purified, and then subjected to limited proteolysis using papain. Stable analogs of intermediate states were generated using AMPPCP as a nonhydrolyzable ATP analog and AlFx as a phosphate analog, following conditions established for Ca2+-ATPase (SERCA1). Characteristic digestion patterns obtained for different analog intermediates show that CopA undergoes domain rearrangements very similar to those of SERCA1. Digestion sites were identified on the loops connecting the A-domain and the transmembrane helices M2 and M3 as well as on that connecting the N-terminal metal binding domain (NMBD) and the first transmembrane helix, Ma. These digestion sites were protected in the E1P.ADP and E2P analogs, whereas the M2-A-domain loop was cleaved specifically in the absence of ions to be transported, just as in SERCA1. ATPase activity was lost when the link between the NMBD and the transmembrane domain was cleaved, indicating that the NMBD plays a critical role in ATP hydrolysis in T. maritima CopA. The change in susceptibility of the loop between the NMBD and Ma helix provides evidence that the NMBD is associated to the A-domain and recruited into domain rearrangements and that the Ma helix is the counterpart of the M1 helix in SERCA1 and Mb and Mc are uniquely inserted before M2.

Importance of Leu99 in Transmembrane Segment M1 of the Na+,K+-ATPase in the Binding and Occlusion of K+

Twenty-six point mutations were introduced into the N-terminal and middle parts of transmembrane segment M1 of the Na+, K+ -ATPase and its cytosolic extension. None of the alterations to charged and polar residues in the N-terminal part of M1 and its cytosolic extension had any major effect on the cation binding properties, thus rejecting the hypothesis that these residues are involved in cation selectivity. By contrast, specific residues in the middle part of M1, particularly Leu(99), were found critical to K+ interaction of the enzyme. Hence, mutation L99A reduced the affinity for K+ activation of E2P dephosphorylation 17-fold, and L99F reduced the equilibrium level of the K+-occluded intermediate [K2]E2 and increased the rate of K+ deocclusion 39-fold, i.e. more than seen for mutation E329Q of the cation-binding glutamate in M4. L99Q affected K+ interaction in yet another way, the equilibrium level of [K2]E2 being slightly increased despite an increased rate of K+ deocclusion, suggesting that the K+ ions leave and enter the occlusion pocket more frequently than in the wild type. L99Q furthermore affected the ability to discriminate between Na+ and K+ on the extracellular side. Our findings can be explained by a structural model in which Leu(99) and Glu(329) interact and cooperate in K+ binding and gating of the K+ sites. The disturbance of K+ interaction in mutants with alteration to Leu(91), Phe(95), Ser(96), or Leu(98) could be a consequence of the roles of these residues in positioning the M1 helix optimally for the interaction between Leu(99) and Glu(329). Phe(95) may serve to stabilize the pivot for movement of M1 through interaction with Ile(287) in M3.

A stepwise mechanism for acetylcholine receptor channel gating

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. We used single-molecule kinetic analysis to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the alpha-subunit of the murine acetylcholine receptor. This analysis produces a 'Phi-value' for a given residue that reflects its open-like versus closed-like character at the transition state. Here we show that most of the residues throughout the length of M2 have a Phi-value of approximately 0.64 but that some near the middle have lower Phi-values of 0.52 or 0.31, suggesting that alphaM2 moves in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.

The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

An Unusual Twin-His Arrangement in the Pore of Ammonia Channels Is Essential for Substrate Conductance

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.

Association of Renal Na,K-ATPase α-Subunit with the β- and γ-Subunits Based on Cryoelectron Microscopy

Na,K-ATPase transports Na(+) and K(+) across cell membranes and consists of alpha- and beta-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 A resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the gamma-subunit. A homology model for the alpha-subunit was calculated from a Ca(2+)-ATPase structure and used to locate the additional beta- and gamma-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the beta-subunit is located close to transmembrane helices M8 and M10 and the gamma-subunit is adjacent to helices M2 and M9 of the alpha-subunit.

The Structural Context of Disease-causing Mutations in Gap Junctions

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.

Kir6.2 Channel Gating by Intracellular Protons: Subunit Stoichiometry for Ligand Binding and Channel Gating

The adenosine triphosphate-sensitive K(+) (K(ATP)) channels are gated by several metabolites, whereas the gating mechanism remains unclear. Kir6.2, a pore-forming subunit of the K(ATP) channels, has all machineries for ligand binding and channel gating. In Kir6.2, His175 is the protonation site and Thr71 and Cys166 are involved in channel gating. Here, we show how individual subunits act in proton binding and channel gating by selectively disrupting functional subunits using these residues. All homomeric dimers and tetramers showed pH sensitivity similar to the monomeric channels. Concatenated construction of wild type with disrupted subunits revealed that none of these residues had a dominant-negative effect on the proton-dependent channel gating. Subunit action in proton binding was almost identical to that for channel gating involving Cys166, suggesting a one-to-one coupling from the C terminus to the M2 helix. This was significantly different from the effect of T71Y heteromultimers, suggesting distinct contributions of M1 and M2 helices to channel gating. Subunits underwent concerted rather than independent action. Two wild-type subunits appeared to act as a functional dimer in both cis and trans configurations. The understanding of K(ATP) channel gating by intracellular pH has a profound impact on cellular responses to metabolic stress as a significant drop in intracellular pH is more frequently seen under a number of physiological and pathophysiological conditions than a sole decrease in intracellular ATP levels.

Surface Structure and Its Dynamic Rearrangements of the KcsA Potassium Channel upon Gating and Tetrabutylammonium Blocking

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.