Dynamic aspects of functioning membrane proteins

The Journal of Physiology Symposium entitled ‘Dynamic aspects of functioning membrane proteins’ was held on 31 July at the 36th International Congress of Physiological Sciences (IUPS2009) in Kyoto. Here we would like to briefly outline the context and scope of the symposium. An intense period of biochemistry and genetics in the 1980s and since led to the cloning of most of the members of the largest ion channel and receptor families and to their extensive molecular and biophysical analyses. Crystal structure analyses have provided us with precise static images and some remarkable insights into functional mechanism. The recent challenge has been to reconstruct the structural rearrangements that underlie functional transitions. The focus of the symposium was on the functional dynamics of ion channels and receptors. The six speakers work on a wide range of membrane proteins, including ion channels and G-protein coupled receptors (GPCR). Their common theme is to combine spectroscopic and structural analysis with functional analysis to probe the structural changes that turn the channels and receptors on and off. The methods include fluorescent resonance energy transfer (FRET), electron spin resonance (ESR), crystallography, voltage clamp fluorometry, single molecule imaging and electrophysiology to determine the structures of wild-type and mutant membrane proteins, work out the composition of protein complexes, and monitor structural rearrangements and correlate them to functional transitions measured at the same time. Four review articles summarizing these presentations are included in this issue. In the review from Kubo's group (Kubo et al. 2009) an unusual dynamic aspect of ATP receptor channel P2X2 is investigated. Ion channel pores are typically fine-tuned for a fixed ion selectivity. However there are certain exceptions to this rule. K+ channels have a form of inactivation that involves a rearrangement of the pore that changes their selectivity. P2X2 channels have an even more radical transition called ‘pore dilatation’, which takes place upon long exposures to ATP and which radically increases the size of objects that can pass through the pore. Kubo's group has found that the properties of the P2X2 pore, such as rectification and ion selectivity, depend on the density of channels on the membrane. This surprising observation suggests a novel regulatory mechanism in which interactions between channels on the membrane alters their pore properties. Moreover, they observed that the P2X2 channel shows voltage-dependent gating even though it has no canonical voltage sensor, indicating that P2X2 is not a simple on–off switch operated by ATP alone. Isacoff's group employed methods that they developed for studying the dynamic conformational changes of voltage-gated K+ channels to study the newest member of the voltage-gated channel superfamily, the Hv1 proton channel (Tombola et al. 2009). Hv1 has a voltage-sensing domain, but no pore domain, leading to the question of where the permeation pathway for H+ lies. Since in many other channels pores lie at the interface between multiple subunits this question led to the need to know the number of subunits in the functional unit of the channel. They used a method recently developed in their lab (Ulbrich & Isacoff, 2007) to count subunits in intact protein complexes on the surface of a cell, using single molecule photo-bleaching under evanescent field microscopy. They found that Hv1 is a dimer. By stripping apart the dimer in chimeric constructs with the distantly related and monomeric Ci-VSP (Kohout et al. 2008), and by using tandemly ligated dimers of WT-WT or WT-214C, where the 214C subunit can be blocked by conjugation with MTSET, they showed that each of the subunits has its own separately gated pore. They further obtained evidence that suggests both where the pore is located and how it may be gated. Bezanilla's group has studied the conformational changes underlying gating of both voltage-gated channels and Ci-VSP, which has VSD and a cytoplasmic phosphatase domain instead of pore region. In the symposium, Bezanilla presented a new application of site-specific quenching of fluorescent probes and lanthanide-based fluorescence resonance energy transfer (LRET), and these optical experiments were complemented by Cys/His scanning mutagenesis of the voltage sensor domain. Taken together with their experimental results and the crystal structure information, they proposed a picture of the conformational changes underlying voltage sensing and gating. They concluded there are at least three major states that are voltage and time dependent and that the conversion between states includes rotation, translocation and tilt changes of the S4 segment (Bezanilla, 2008; Villalba-Galea et al. 2008). Perozo's group has approached the conformational change of the pH sensitive K+ channel KcsA and the stretch activated channel MscS (Vasquez et al. 2008) by combining specroscopic techniques such as ESR and nuclear magnetic resonance (NMR), X-ray crystallography and electrophysiological methods. In the symposium, Perozo presented an astonishing set of multiple crystal structures of KcsA trapped in multiple conformations including open/conductive, open/inactivated and several partially open intermediates (Uysal et al. 2009). Based on these data and the functional spectroscopic and computational data, a molecular basis of activation and inactivation gating of KcsA channel was proposed in which the inner bundle gate and the selectivity filter are coupled. Reuveny's group has been using FRET to study the molecular rearrangements during the gating of G-protein coupled inwardly rectifying K+ channels (GIRK) and obtained some unexpected results as follows. GIRK is an effecter of G protein, which is activated by the Gβγ subunit. The Reuveny group found that, remarkably, Gβγ subunits are already situated in close proximity to GIRK even before ligand activation of the GPCR. They then found that Gβγ translocates from one site to another on the GIRK channel upon activation (Raveh et al. 2009). Pin's group has been working on the structural rearrangements and the signalling mechanisms of the metabotropic glutamate receptor (mGluR) and the GABAB receptor, members of the family-C G-protein coupled receptor family. They showed that dimerization is required for the function of mGluR and the GABAB receptors and derived constraints for relative movement between the subunits that occur during activation. They also showed that, in contrast to mGluR, which is a strict dimer, the GABAB receptor forms a larger complex both in the heterologous expression system and in the brain. As the coupling efficiency of the larger complex with G proteins was lower than with dimers, higher assembly was postulated to increase the functional diversity (Pin et al. 2009). The six presentations provided an update on the use of modern methods for probing the function, structure and dynamics of membrane signalling proteins and insights into functional principles in several specific channels and receptors.