The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.Many membrane proteins form multimers before they achieve a functional state and are transported to the plasma membrane of the cell. The stoichiometry of a complex is precisely regulated and is fundamental to its functional properties. Subunit stoichiometry is usually assessed via bulk biochemical and macroscopic functional analyses. For example, the multimeric state of K + channels in the squid giant axon was originally predicted from the shape of the current trace during opening of the channels in voltage clamp 1 . A kinetic analysis of inactivation 2 and an analysis of currents in mixed subunit channels 3 indicated that the channels are probably composed of four similar or identical subunits, which was later confirmed by crystallography 4 . Sometimes macroscopic functional recordings do not distinguish stoichiometry precisely. For example, CNG channels for years had been assumed to be composed of a 2:2 stoichiometry of CNGA1 and CNGB1 subunits 5 , but has been shown to actually be composed of a 3:1 CNGA1 to CNGB1 stoichiometry by biochemical analysis 6 and using fluorescence energy resonance transfer between fluorescent proteins fused to CNGA1 and CNGB1 ( ref. 7 ).The well-studied glutamate-gated NMDA receptors are tetramers containing two NR1 and two NR2 subunits. This stoichiometry had been deduced from macroscopic functional analysis, in which coexpression of wild-type and mutant subunits resulted in a triphasic response to agonists that could be explained by mixture of receptors containing zero, one or two mutant NR1 or NR2 subunits 8 , and from single-channel recordings that showed distinct behaviors consistent with the possible mixed stoichiometries 9 , and confirmed by crystallography 10 . Less is known about NMDA receptors containing the more recently discovered NR3 subunit, which is thought to be involved in synaptic development 11 . Initially it was thought that NR3 coassembles with NR1 and NR2 to form glutamate-gated receptors with unique properties 11 . More recently, and quite surprisingly, the NR3 subunit ligand binding domain had been found to bind glycine and not glutamate, and

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed co-receptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into use of IRs as genetically encoded chemical sensors.

We have probed internal and external accessibility of S4 residues to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethlammonium (MTSET) in both open and closed, cysteine-substituted Shaker K+ channels. Our results indicate that S4 traverses the membrane with no more than 5 amino acids in the closed state, and that the distribution of buried residues changes when channels open. This change argues for a displacement of S4 through the plane of the membrane in which an initially intracellular residue moves to within 3 amino acids of the extracellular solution. These results demonstrate that the putative voltage-sensing charges of S4 actually reside in the membrane and that they move outward when channels open. We consider constraints placed on channel structure by these results.

Neurexins are a large family of proteins that act as neuronal cell-surface receptors. The function and localization of the various neurexins, however, have not yet been clarified. Beta-neurexins are candidate receptors for neuroligin-1, a postsynaptic membrane protein that can trigger synapse formation at axon contacts. Here we report that neurexins are concentrated at synapses and that purified neuroligin is sufficient to cluster neurexin and to induce presynaptic differentiation. Oligomerization of neuroligin is required for its function, and we find that beta-neurexin clustering is sufficient to trigger the recruitment of synaptic vesicles through interactions that require the cytoplasmic domain of neurexin. We propose a two-step model in which postsynaptic neuroligin multimers initially cluster axonal neurexins. In response to this clustering, neurexins nucleate the assembly of a cytoplasmic scaffold to which the exocytotic apparatus is recruited.

Neurons have ion channels that are directly gated by voltage, ligands and temperature but not by light. Using structure-based design, we have developed a new chemical gate that confers light sensitivity to an ion channel. The gate includes a functional group for selective conjugation to an engineered K + channel, a pore blocker and a photoisomerizable azobenzene. Long-wavelength light drives the azobenzene moiety into its extended trans configuration, allowing the blocker to reach the pore. Short-wavelength light generates the shorter cis configuration, retracting the blocker and allowing conduction. Exogenous expression of these channels in rat hippocampal neurons, followed by chemical modification with the photoswitchable gate, enables different wavelengths of light to switch action potential firing on and off. These synthetic photoisomerizable azobenzene-regulated K + (SPARK) channels allow rapid, precise and reversible control over neuronal firing, with potential applications for dissecting neural circuits and controlling activity downstream from sites of neural damage or degeneration.Natural photoreceptive proteins, such as rhodopsin, have a covalently attached chromophore that directly activates the protein on exposure to light. Several strategies have been used to enable light regulation of proteins that are not intrinsically light sensitive. Light can be used indirectly to activate receptor proteins, for example, by making a ligand available from a caged precursor 1 . Light can also be used to directly photoisomerize a synthetic molecule that is covalently attached to a protein, thereby imposing conformational changes 2,3 . We have combined these ideas by synthesizing a photoisomerizable tether that attaches a specific ligand on a protein near its normal binding site. Photoswitching of the tethered ligand rapidly regulates its ability to reach its binding site, thereby switching the protein on and off without causing unnatural conformational changes. Photoswitchable tethered ligands can be agonists (for example for nicotinic acetylcholine receptors 4 ), antagonists, or other regulators of protein function. We have applied this general design principle to an ion channel, where the chosen ligand is a blocker of the channel's pore. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diffusion kinetics. Recently, the exogenous expression of genes encoding ion channels has been used to influence electrical activity in specific neurons 7-9 , but the onset and reversal of gene expression is slow. A modification of this technique, in which a receptor from one species is introduced into another that lacks a natural ligand, dramatically improves temporal control 10,11 , but this approach still relies on a diffusible ligand whose time course of removal limits reversibility. Finally, photic regulation has been conferred on neurons by introducing a rhodopsin-based signal transduction cascade 12 . This technique requires coordinated exogenous expression of three different genes and produ...

In response to membrane depolarization, voltage-gated ion channels undergo a structural rearrangement that moves charges or dipoles in the membrane electric field and opens the channel-conducting pathway. By combination of site-specific fluorescent labeling of the Shaker potassium channel protein with voltage clamping, this gating conformational change was measured in real time. During channel activation, a stretch of at least seven amino acids of the putative transmembrane segment S4 moved from a buried position into the extracellular environment. This movement correlated with the displacement of the gating charge, providing physical evidence in support of the hypothesis that S4 is the voltage sensor of voltage-gated ion channels.

The precise regulation of protein activity is fundamental to life. The allosteric control of an active site by a remote regulatory binding site is a mechanism of regulation found across protein classes, from enzymes to motors to signaling proteins. We describe a general approach for manipulating allosteric control using synthetic optical switches. Our strategy is exemplified by a ligand-gated ion channel of central importance in neuroscience, the ionotropic glutamate receptor (iGluR). Using structure-based design, we have modified its ubiquitous clamshell-type ligand-binding domain to develop a light-activated channel, which we call LiGluR. An agonist is covalently tethered to the protein through an azobenzene moiety, which functions as the optical switch. The agonist is reversibly presented to the binding site upon photoisomerization, initiating clamshell domain closure and concomitant channel gating. Photoswitching occurs on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength. Our device has potential uses not only in biology but also in bioelectronics and nanotechnology.Many proteins function like molecular machines that undergo mechanical movements in response to input signals. These signals can consist of changes in voltage, membrane tension, temperature or, most commonly, ligand concentration. Ligands provide information about events in the external world or about the energetic or biosynthetic state of the cell. They can be as small as a proton or as large as a whole protein. In allostery, ligand binding induces a structural change of a sensor domain, which propagates to a functional domain of the protein and alters its behavior. Such conformational control can operate over long distances, crossing a membrane or passing from one protein to another in a complex.Reengineering of nanoscopic protein machines to contain artificial control elements would be a major benefit for biology and technology. Optical switches would be especially powerful, as they could be activated remotely with precise temporal and spatial control 1,2 . A simple design Correspondence should be addressed to E.I. (ehud@berkeley.edu) and D.T. (trauner@berkeley.edu).. 5 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Chemical Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strategy would be to modify a protein by attaching a synthetic ligand whose binding ability could be altered by light. These ligands tethered via an optical switch could function in two ways. They could conditionally block the active site of an enzyme or the pore of a channel without inducing major conformational changes in the protein, or they could reversibly present an agonist to an allosteric binding site and conditionally trigger the normal conformational changes of ac...

We present the synthesis, properties, and biological applications of Coppersensor-1 (CS1), a new water-soluble, turn-on fluorescent sensor for intracellular imaging of copper in living biological samples. CS1 utilizes a BODIPY reporter and thioether-rich receptor to provide high selectivity and sensitivity for Cu+ over other biologically relevant metal ions, including Cu2+, in aqueous solution. This BODIPY-based probe is the first Cu+-responsive sensor with visible excitation and emission profiles and gives a 10-fold turn-on response for detecting this ion. Confocal microscopy experiments further establish that CS1 is membrane-permeable and can successfully monitor intracellular Cu+ levels within living cells.