Ehud Y. Isacoff

Direct physical measure of conformational rearrangement underlying potassium channel gating

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.

Neurexin mediates the assembly of presynaptic terminals

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.

Light-activated ion channels for remote control of neuronal firing

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.

Transmembrane Movement of the Shaker K+ Channel S4

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.

Subunit counting in membrane-bound proteins

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.

A Genetically Encoded Optical Probe of Membrane Voltage

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed a novel, genetically encoded probe that can be used to measure transmembrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive K+ channel so that voltage-dependent rearrangements in the K+ channel would induce changes in the fluorescence of GFP. The probe has a maximal fractional fluorescence change of 5.1%, making it comparable to some of the best organic voltage-sensitive dyes. Moreover, the fluorescent signal is expanded in time in a way that makes the signal 30-fold easier to detect. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism noninvasively and targeted to specific developmental stages, brain regions, cell types, and subcellular compartments.

Analysis of a RanGTP-regulated gradient in mitotic somatic cells

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-β family. The Ran–importin-β system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran–importin-β system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-β by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran–importin-β–cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

Optogenetic dissection of a behavioural module in the vertebrate spinal cord.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer–Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer–Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Remote Control of Neuronal Activity with a Light-Gated Glutamate Receptor

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.

Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing it is necessary to define the structure and motion of the S4 segment, the portion of each voltage-sensing domain that moves charged residues across the membrane in response to voltage change. We have addressed this problem by using fluorescence resonance energy transfer as a spectroscopic ruler to determine distances between S4s in the Shaker K+ channel in different gating states. Here we provide evidence consistent with S4 being a tilted helix that twists during activation. We propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that it is involved in coupling voltage sensing to gating.