Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold.

TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.

Internal Proton Transfer in the Activation of Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length of side chains determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.

High-speed scanless entire bandwidth mid-infrared chemical imaging.

Mid-infrared spectroscopy probes molecular vibrations to identify chemical species and functional groups. Therefore, mid-infrared hyperspectral imaging is one of the most powerful and promising candidates for chemical imaging using optical methods. Yet high-speed and entire bandwidth mid-infrared hyperspectral imaging has not been realized. Here we report a mid-infrared hyperspectral chemical imaging technique that uses chirped pulse upconversion of sub-cycle pulses at the image plane. This technique offers a lateral resolution of 15 µm, and the field of view is adjustable between 800 µm × 600 µm to 12 mm × 9 mm. The hyperspectral imaging produces a 640 × 480 pixel image in 8 s, which covers a spectral range of 640-3015 cm-1, comprising 1069 wavelength points and offering a wavenumber resolution of 2.6-3.7 cm-1. For discrete frequency mid-infrared imaging, the measurement speed reaches a frame rate of 5 kHz, the repetition rate of the laser. As a demonstration, we effectively identified and mapped different components in a microfluidic device, plant cell, and mouse embryo section. The great capacity and latent force of this technique in chemical imaging promise to be applied to many fields such as chemical analysis, biology, and medicine.

Protonation of Asp116 and distortion of the all-trans retinal chromophore in Krokinobacter eikastus rhodopsin 2 causes a redshift in absorption maximum upon dehydration.

Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins. It was reported that a sodium-pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2), showed a color change from red to purple upon dehydration under crystalline conditions. Here, we applied comprehensive visible and IR absorption spectroscopy and resonance Raman spectroscopy to KR2 in liposomes under hydration-controlled conditions. A large increase in the hydrogen-out-of-plane (HOOP) vibration at 947 (H-C11=C12-H Au mode) and moderate increases at 893 (C7-H and C10-H) and 808 (C14-H) cm-1 were observed under dehydrated conditions, which were assigned by using systematically deuterated retinal. Moreover, the Asn variant at Asp116, which functions as a counter ion for the protonated retinal Schiff base (PRSB), caused a large redshift in the absorption maximum and constitutive increase in the HOOP modes under hydrated and dehydrated conditions. The protonation of a counter ion at Asp116 clearly causes a redshift in the absorption maximum as the all-trans retinal chromophore twists upon dehydration. Namely, the results strongly suggested that water molecules are important for maintaining the hydrogen-bonding network at the PRSB and deprotonation state of Asp116 in KR2.

Covalent Bond between the Lys-255 Residue and the Main Chain Is Responsible for Stable Retinal Chromophore Binding and Sodium-Pumping Activity of Krokinobacter Rhodopsin 2.

Microbial rhodopsins are light-receptive proteins with various functions triggered by the photoisomerization of the retinal chromophore from the all-trans to 13-cis configuration. A retinal chromophore is covalently bound to a lysine residue in the middle of the seventh transmembrane helix via a protonated Schiff base. Bacteriorhodopsin (BR) variants lacking a covalent bond between the side chain of Lys-216 and the main chain formed purple pigments and exhibited a proton-pumping function. Therefore, the covalent bond linking the lysine residue and the protein backbone is not considered a prerequisite for microbial rhodopsin function. To further examine this hypothesis regarding the role of the covalent bond at the lysine side chain for rhodopsin functions, we investigated K255G and K255A variants of sodium-pumping rhodopsin, Krokinobacter rhodopsin 2 (KR2), with an alkylamine retinal Schiff base (prepared by mixing ethyl- or n-propylamine and retinal (EtSB or nPrSB)). The KR2 K255G variant incorporated nPrSB and EtSB as similarly to the BR variants, whereas the K255A variant did not incorporate these alkylamine Schiff bases. The absorption maximum of K255G + nPrSB was 524-516 nm, which was close to the 526 nm absorption maximum of the wild-type + all-trans retinal (ATR). However, the K255G + nPrSB did not exhibit any ion transport activity. Since the KR2 K255G variant easily released nPrSB during light illumination and did not form an O intermediate, we concluded that a covalent bond at Lys-255 is important for the stable binding of the retinal chromophore and formation of an O intermediate to achieve light-driven Na+ pump function in KR2.

Concerted primary proton transfer reactions in a thermophilic rhodopsin studied by time-resolved infrared spectroscopy at high temperature.

The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to ß-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to ß-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin.

Specific zinc binding to heliorhodopsin.

Heliorhodopsins (HeRs), a recently discovered family of rhodopsins, have an inverted membrane topology compared to animal and microbial rhodopsins. The slow photocycle of HeRs suggests a light-sensor function, although the actual function remains unknown. Although HeRs exhibit no specific binding of monovalent cations or anions, recent ATR-FTIR spectroscopy studies have demonstrated the binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR) and 48C12. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding, suggesting a possible modification of the HeR function by Zn2+. The present study shows that Zn2+-binding lowers the thermal stability of TaHeR, and slows back proton transfer to the retinal Schiff base (M decay) during its photocycle. Zn2+-binding was similarly observed for a TaHeR opsin that lacks the retinal chromophore. We then studied the Zn2+-binding site by means of the ATR-FTIR spectroscopy of site-directed mutants. Among five and four mutants of His and Asp/Glu, respectively, only E150Q exhibited a completely different spectral feature of the α-helix (amide-I) in ATR-FTIR spectroscopy, suggesting that E150 is responsible for Zn2+-binding. Molecular dynamics (MD) simulations built a coordination structure of Zn2+-bound TaHeR, where E150 and protein bound water molecules participate in direct coordination. It was concluded that the specific binding site of Zn2+ is located at the cytoplasmic side of TaHeR, and that Zn2+-binding affects the structure and structural dynamics, possibly modifying the unknown function of TaHeR.

Unusual Photoisomerization Pathway in a Near-Infrared Light Absorbing Enzymerhodopsin.

Microbial and animal rhodopsins possess retinal chromophores which capture light and normally photoisomerize from all-trans to 13-cis and from 11-cis to all-trans-retinal, respectively. Here, we show that a near-infrared light-absorbing enzymerhodopsin from Obelidium mucronatum (OmNeoR) contains the all-trans form in the dark but isomerizes into the 7-cis form upon illumination. The photoproduct (λmax = 372 nm; P372) possesses a deprotonated Schiff base, and the system exhibits a bistable nature. The photochemistry of OmNeoR was arrested at <270 K, indicating the presence of a potential barrier in the excited state. Formation of P372 is accompanied by protonation changes of protonated carboxylic acids and peptide backbone changes of an α-helix. Photoisomerization from the all-trans to 7-cis retinal conformation rarely occurs in any solvent and protein environments; thus, the present study reports on a novel photochemistry mediated by a microbial rhodopsin, leading from the all-trans to 7-cis form selectively.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade.

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12.

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) α-helices and an all-trans-retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins. The X-ray crystal structure of HeR 48C12 was elucidated after the first report on a HeR variant from Thermoplasmatales archaeon SG8-52-1, which revealed the water-mediated hydrogen-bonding network connected to the Schiff base region in the cytoplasmic side. Herein, low-temperature light-induced FTIR spectroscopic analyses of HeR 48C12 and 15N isotopically labeled proteins were used to elucidate the structural changes during retinal photoisomerization. N-D stretching vibrations of the protonated retinal Schiff base (PRSB) at 2286 and 2302 cm-1 in the dark state, and 2239 and 2252 cm-1 in the K intermediate were observed. The frequency changes indicated that the hydrogen bond of PRSB strengthens upon photoisomerization in HeR. Moreover, O-D stretching vibration frequencies of the internal water molecules indicate that the hydrogen-bonding strength decreases concomitantly. Therefore, the PRSB hydrogen bond responds to photoisomerization in an opposite way to the hydrogen-bonding network involving water molecules. No frequency changes of the indole N-H or N-D stretching vibrations of tryptophan residues were observed upon photoisomerization, suggesting that all tryptophan residues in the HeR 48C12 maintained the hydrogen-bonding strengths in the K intermediate. These results provide insights into the molecular mechanism of the energy storage and propagation upon retinal photoisomerization in HeR.

Optogenetic Modulation of Ion Channels by Photoreceptive Proteins.

In these 15 years, researches to control cellular responses by light have flourished dramatically to establish "optogenetics" as a research field. In particular, light-dependent excitation/inhibition of neural cells using channelrhodopsins or other microbial rhodopsins is the most powerful and the most widely used optogenetic technique. New channelrhodopsin-based optogenetic tools having favorable characteristics have been identified from a wide variety of organisms or created through mutagenesis. Despite the great efforts, some neuronal activities are still hard to be manipulated by the channelrhodopsin-based tools, indicating that complementary approaches are needed to make optogenetics more comprehensive. One of the feasible and complementary approaches is optical control of ion channels using photoreceptive proteins other than channelrhodopsins. In particular, animal opsins can modulate various ion channels via light-dependent G protein activation. In this chapter, we summarize how such alternative optogenetic tools work and they will be improved.

Zinc Binding to Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, has an inverted membrane topology compared to animal and microbial rhodopsins, and no ion-transport activity. The slow photocycle of HeRs suggests a light-sensor function, although the function remains unknown. HeRs exhibit no specific binding of monovalent cations or anions. Despite this, ATR-FTIR spectroscopy in the present study demonstrates binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR). The biding of Zn2+ to 0.2 mM Kd is accompanied by helical structural perturbations without altering its color. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding. Similar results were obtained for HeR 48C12. These findings suggest a possible modification of HeR function by Zn2+.

Mechanism of Inward Proton Transport in an Antarctic Microbial Rhodopsin.

Although the outward-directed proton transport across biological membranes is well studied and its importance for bioenergetics is clearly understood, inward-directed light-driven proton pumping by microbial rhodopsins has remained a mystery both physiologically and mechanistically. A new family of Antarctic rhodopsins, which is a subgroup within a novel class of schizorhodopsins reported recently, includes a member, denoted as AntR, which proved amenable to extensive characterization with experiments and computation. Phylogenetic analyses identify AntR as distinct from the well-studied microbial rhodopsins that function as outward-directed ion pumps, and bioinformatics sequence analyses reveal amino acid substitutions at conserved sites essential for outward proton pumping. Modeling and numerical simulations of AntR, combined with advanced analyses using the graph theory and centrality measures from social sciences, identify the dynamic three-dimensional network of hydrogen-bonded water molecules and amino acid residues that function as communication hubs in AntR. This network undergoes major rearrangement upon retinal isomerization, showing important changes in the connectivity of the active center, retinal Schiff base, to the opposing sides of the membrane, as required for proton transport. Numerical simulations and experimental studies of the photochemical cycle of AntR by spectroscopy and site-directed mutagenesis allowed us to identify pathways that could conduct protons in the direction opposite to that commonly known for outward-directed pumps.

Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2.

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm-1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm-1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.

Regulation of Photocycle Kinetics of Photoactive Yellow Protein by Modulating Flexibility of the ß-Turn.

The role of the significant flexibility of the ß-turn in photoactive yellow protein (PYP) due to Gly115 was studied. G115A and G115P mutations were observed to accelerate the photocycle and shift the equilibrium between the late photocycle intermediate (pB) and its precursor (pR) toward pR. Thermodynamic analysis of dark-state recovery from pB demonstrated that the transition state (pB⧧) has a negative change in transition heat capacity, suggesting that an exposed hydrophobic surface of pB is buried in pB⧧. Fourier transform infrared spectroscopy showed that the structural ensemble of pB is populated by the compact structure in G115P. Taken together, the rigid structure induced by mutation of Gly115 facilitates its transition to pB⧧, which adopts a substantially more compact structure as opposed to the ensemble-averaged structure of pB. The photocycle kinetics of PYP may be fine-tuned by modulating the flexibility of the 115 loop to activate an appropriate number of transducer proteins.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Vibrational and Molecular Properties of Mg2+ Binding and Ion Selectivity in the Magnesium Channel MgtE.

Magnesium ions (Mg2+) are crucial for various biological processes. A bacterial Mg2+ channel, MgtE, tightly regulates the intracellular Mg2+ concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane α helices forming a central pore, and intracellular soluble domains constituting a Mg2+ sensor. The ion selectivity for Mg2+ over Ca2+ resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg2+ and Ca2+. Using site-directed mutagenesis, vibrational bands at 1421 (Mg2+), 1407 (Mg2+), ∼1440 (Ca2+), and 1390 (Ca2+) cm-1 were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of the Mg2+-sensitive carboxylate bands, suggesting a highly optimized geometry for accommodating a Mg2+ ion. The dependency of the vibrational changes on Mg2+ and Ca2+ concentrations revealed the presence of a two different classes of binding sites: a high affinity site for Mg2+ ( Kd ≈ 0.3 mM) with low Ca2+ affinity ( Kd ≈ 80 mM), and a medium affinity site for Mg2+ ( Kd ≈ 2 mM) and Ca2+ ( Kd ≈ 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively. With the aid of molecular dynamics simulation and normal-mode analysis by quantum chemistry, we confirm that changes in carboxylate bands of the high affinity binding site originate from Asp432 in the central cavity.

Structural properties determining low K+ affinity of the selectivity filter in the TWIK1 K+ channel.

Canonical K+ channels are tetrameric and highly K+-selective, whereas two-pore-domain K+ (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na+ and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K+-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K+ ions, WT and both variants exhibit an amide-I band at 1680 cm-1 This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K+, a feature very similar to that of the canonical K+ channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K+ affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm-1 band upon changes from K+ to Rb+ or Cs+ conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K+ and Na+ solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K+ channels.