NIPS–JP symposium: Cutting‐edge approaches towards the functioning mechanisms of membrane proteins

The 45th joint symposium of The National Institute for Physiological Sciences (NIPS) and The Journal of Physiology (JP) entitled Cutting-edge approaches towards the functioning mechanisms of membrane proteins was held from 25th to 28th Nov 2014 in Okazaki, Japan. Twenty-six speakers, including 10 invited overseas speakers (UK 1, USA 4, Canada 2, Australia 1, France 1, Israel 1), presented recent research achievements. There were also 36 poster presentations. The number of participants was 106 in total. Although cDNA cloning, mutagenesis studies and crystal structure analyses have enabled significant advances in membrane protein research, many questions about functioning mechanisms remain unanswered. Four important topics were covered in the symposium: (1) Dynamic imaging of functioning membrane proteins. This included: single molecule motion analysis by tracing the diffraction pattern of a gold crystal label attached to a channel protein, high speed atomic force microscopy analysis of structural rearrangements of recombinant proteins, visualization of the rotation of a single motor protein, crystal structure analysis of membrane proteins in multiple states, NMR analysis of membrane proteins in multiple states, structural rearrangement analysis by voltage clamp fluorometry and fluorescence resonance energy transfer recording. (2) Novel regulatory mechanisms involving the local environment. Topics covered included expression density-dependent functional changes, external pH-dependent ion selectivity changes, mechanisms of partial agonism in G protein coupled receptor signalling, and functional regulation of cardiac and vascular ion channels by unsaturated fatty acids, sex hormones or oxygen. (3) Understanding structure and function of membrane protein complexes. Examples of topics covered are: single molecule imaging to determine the stoichiometry of the molecular complexes, and functional interactions of co-expressed membrane proteins. (4) The final category concerned aspects of systems physiology, pathophysiology and translational or clinical research. We also learned about the molecular mechanisms of damage of wine flavour by cork taint and fine drug design based on detailed molecular structure. This issue contains six reviews related to the presentations in the symposium. The first from Oiki and colleagues is representative of the post-crystallographic age which focuses on the dynamic aspects of membrane proteins. The key concepts are reconstitution and use of dynamic information to reanimate the crystal structure. They analysed the pH-dependent gating of the KcsA channel at the single molecule level by applying a diffracted X-ray tracking method, and the clustering and dispersion associated with the gating of KcsA by high speed atomic force microscopy. They also performed experiments to analyse the influence of constituents of the lipid bilayer on the function of KcsA (Oiki, 2015). Vandenberg and his colleagues have been working on the biophysical mechanisms of the characteristic gating of the hERG K+ channel and its physiological relevance in cardiac function. They graphically explored inactivation using Rate Equilibrium Free Energy Relationship/ Φ-value analysis. This is based on relationships of free energy changes in kinetics and in thermodynamics induced by perturbations. They introduced single mutations in various regions of the hERG channel and analysed the Φ-values. Through this approach, they describe how the conformational changes associated with inactivation propagate in the hERG channel. They also simulate the cardiac action potential using the O'Hare–Rudy model to show how a decrease in the activity of hERG can result in cardiac arrhythmia (Perry et al. 2015). Lesage and colleagues have been engaged in studying various aspects of K2P K+ channels, including novel regulatory mechanisms such as external pH-dependent dynamic ion selectivity changes in the TWIK1 channel. Their comprehensive review starts with structure and electrophysiological properties, and then describes regulatory mechanisms, heteromultimeric assembly and modification by associated proteins. They also summarize the physiological relevance of K2P channels to human pathophysiology (Feliciangeli et al. 2015). The Larsson group focuses on the characteristic gating of KCNQ1 and the modulation by accessary subunits, KCNE1 and KCNE3. They simultaneously analysed the movement of the voltage sensor and pore opening using voltage clamp fluorometry. In this review, they consider differences of KCNQ1 channel current in various tissues and their physiological relevance, highlighting the contribution of KCNEs. They then describe the general mechanisms of electro-chemical coupling based on the structural model, and discuss the biophysical mechanisms of slow activation of the KCNQ1–KCNE1 channel complex using a state diagram incorporating the results of voltage clamp fluorometry (Liin et al. 2015). Nakajo and Kubo have also been working on the KCNQ1–KCNE1 channel complex. Their review focuses on how KCNE1 binds and alters the function of KCNQ1 channels, describing the effect on the voltage-dependent gating of a possible collision of two bulky amino acids in the S4 and S5 segments of KCNQ1, which occurs only in the presence of KCNE1. They also describe the different sites of interaction with KCNQ1 for KCNE1 and KCNE3, based on their comparative studies (Nakajo & Kubo, 2015). Kurata and colleagues reviewed their own atomic scale structure–function study using a strategy to incorporate unnatural amino acids into the ion channel protein. Based on the crystal structure information, introduction of artificially designed unnatural amino acids into the channel protein provides important insights into functional mechanisms at the atomic scale. The use of unnatural amino acid allows novel changes in the protein structure by design, allowing subtle differences to be introduced to the same position of a channel protein and subsequent analysis of the changes in the biophysical properties. They also describe the effectiveness of this approach for sophisticated next-generation drug design which enables fine tuning of channel function (Pless et al. 2015). We hope these reviews will help to highlight recent developments in current knowledge of the functioning mechanisms of membrane proteins and indicate future directions for this rapidly moving research field.

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