Biology subjects

Isacoff, E. Y.

Publications and source records attributed to Isacoff, E. Y..

11 recordsLinked to original sources

bioRxiv2024

Synapse-to-synapse plasticity variability balanced to generate input-wide constancy of transmitter release

Basal synaptic strength can vary greatly between synapses formed by an individual neuron because of diverse probabilities of action potential (AP) evoked transmitter release (Pr). Optical quantal analysis on large numbers of identified Drosophila larval glutamatergic synapses shows that short-term plasticity (STP) also varies greatly between synapses made by an individual type I motor neuron (MN) onto a single body wall muscle. Synapses with high and low Prand different forms and level of STP have a random spatial distribution in the MN nerve terminal, and ones with very different properties can be located within 200 nm of one other. While synapses start off with widely diverse basal Pr at low MN AP firing frequency and change Prdifferentially when MN firing frequency increases, the overall distribution of Pr remains remarkably constant due to a balance between the numbers of synapses that facilitate and depress as well as their degree of change and basal synaptic weights. This constancy in transmitter release can ensure robustness across changing behavioral conditions.

neuroscience↗

bioRxiv2024

Deficiency in transmitter release triggers homeostatic transcriptional changes that increase presynaptic excitability

Weakening of synaptic transmission at the Drosophila larval neuromuscular junction triggers two forms of homeostatic compensation, one that increases the probability of glutamate release per action potential (Pr) and another that increases motoneuron (MN) activity. We investigated the molecular changes in MNs that underlie the increase in MN activity. RNA-seq analysis on MNs whose glutamate release is weakened by knockdown of components of the MN transmitter release machinery reveals a reduction in expression of a group of genes that encode potassium channels and their positive modulators. These results identify a mechanism of compensation for weakened synaptic transmission by MNs, which engages a transcriptional program in those cells to increase firing and, thereby, ensure sufficient locomotory drive.

neuroscience↗

bioRxiv2024

Circuit firing homeostasis following synaptic perturbation ensures robust behavior

Homeostatic regulation of excitability and synaptic transmission ensures stable neural circuit output under changing conditions. We find that pre- or postsynaptic weakening of motor neuron (MN) to muscle glutamatergic transmission in Drosophila larva has little impact on locomotion, suggesting non-synaptic compensatory mechanisms. In vivo imaging of MN to muscle synaptic transmission and MN activity both show that synaptic weakening increases activity in tonic type Ib MNs, but not in the phasic type Is MN that innervate the same muscles. Additionally, an inhibitory class of pre-MNs that innervates type Ib--but not Is--MNs decreases activity. Our experiments suggest that weakening of MN evoked synaptic release onto the muscle is compensated for by an increase in MN firing due to a combined cell-autonomous increase in excitability and decreased inhibitory central drive. Selectivity for type Ib MNs may serve to restore tonic drive while absence of firing adjustment in the convergent Is MN can maintain the contraction wave dynamics needed for locomotion.

neuroscience↗

bioRxiv2024

Molecular insights into G protein specificity and biased agonism at the β2-adrenergic receptor

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The {beta}2-adrenergic receptor is an example of GPCR with high selectivity for Gs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gi family of G proteins inhibiting adenylyl cyclase. By developing a Gi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gs and Gi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

molecular biology↗

bioRxiv2024

Domain coupling in activation of a family C GPCR

The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function. The molecular basis for this diversity remains poorly delineated. We employ molecular dynamics, single-molecule spectroscopy, and hydrogen-deuterium exchange to dissect the pathway of activation triggered by glutamate. We find that activation entails multiple loosely coupled steps and identify a novel pre-active intermediate whose transition to the active state forms dimer interactions that set signaling efficacy. Such subunit interactions generate functional diversity that differs across homodimers and heterodimers. The agonist-bound receptor is remarkably dynamic, with low occupancy of G protein-coupling conformations, providing considerable headroom for modulation of the landscape by allosteric ligands. Sites of sequence diversity within the dimerization interface and diverse coupling between activation rearrangements may contribute to precise decoding of glutamate signals and transients over broad spatial and temporal scales.

biophysics↗

bioRxiv2024

Conformational basis of subtype-specific allosteric control of NMDA receptor gating

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that are integral to synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties, which support their individual physiological roles in the nervous system. To understand the conformational basis of this diversity, we assessed the conformation of the common GluN1 subunit in receptors with different GluN2 subunits using single-molecule fluorescence resonance energy transfer (smFRET). We established smFRET sensors in the ligand binding domain and modulatory amino-terminal domain to study an apo-like state and partially liganded activation intermediates, which have been elusive to structural analysis. Our results demonstrate a strong, subtype- specific influence of apo and glutamate-bound GluN2 subunits on GluN1 rearrangements, suggesting a conformational basis for the highly divergent levels of receptor activity, desensitization and agonist potency. Chimeric analysis reveals structural determinants that contribute to the subtype differences. Our study provides a framework for understanding GluN2-dependent functional properties and could open new avenues for subtype-specific modulation.

biophysics↗

bioRxiv2023

Development of covalent chemogenetic K2P channel activators

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (Covalent Activation of TREK family K+ channels to cLAmp Membrane Potential) that leverages the discovery of a site in the K2P modulator pocket that reacts with electrophile-bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2P channel activity that should facilitate studies both molecular and systems level studies of K2P function and enable the search for new K2P modulators.

biophysics↗

bioRxiv2023

Step-wise activation of a Family C GPCR

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain (ECD) that is linked via a cysteine-rich domain (CRDs) to their 7-transmembrane (TM) domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the ECD to the G protein-coupling TM. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

biophysics↗

bioRxiv2022

Negative allosteric modulation of the glucagon receptor by RAMP2

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrates that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 [A] resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

biochemistry↗

bioRxiv2022

Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels

Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (Swell1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents implicated in a broad range of physiology. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here, we leverage a fiducial-tagging strategy to determine single-particle cryo-electron microscopy structures of heterohexameric LRRC8A:C channels in detergent micelles and lipid nanodiscs in three conformations. LRRC8A:C channels show pronounced changes in channel architecture compared to homomeric channels due to heterotypic cytoplasmic LRR interactions that displace LRRs and the LRRC8C subunit away from the conduction axis and poise the channel for activation. The structures and associated functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. Together, our results provide insight into determinants for heteromeric LRRC8 channel assembly, activity, and gating by lipids.

biophysics↗

bioRxiv2022

Restoration of high-sensitivity patterned vision in motion with an engineered light-gated G protein-coupled receptor

Inherited retinal degenerations (IRDs) result in blindness due to apoptotic cell death of rods and cones, but spare other retinal neurons, providing a potential that delivery of a light-activated signaling protein to surviving neurons may restore vision. We previously demonstrated that aspects of vision could be restored by introduction into surviving cells of a G protein-coupled receptor for glutamate (mGluR) bearing a tethered photoswitchable agonist. However, this system, containing one photoswitchable agonist per glutamate binding site, yielded low sensitivity, responding only to visual stimuli at the intensity of bright outdoor light, similar to channelrhodopsins. To increase sensitivity, we designed a multi-branched photoswitch, bearing four light-activatable glutamates for each glutamate binding site. When tethered to a modified mGluR2 expressed in retinal ganglion cells via intravitreal AAV gene delivery, this photoswitch boosted sensitivity by ~100-fold compared to the unbranched (single photo-ligand) photoswitch. This improvement in sensitivity enabled an IRD mouse model (rd1) to perform visually-guided object recognition under incidental room light and pattern recognition using standard LCD computer displays. The restored line pattern differentiation approached the acuity reported for normal mouse vision. Pattern recognition functioned as well as wildtype vision with line patterns moving at speeds of up to 36{degrees}/s. In summary, this two-component chemical-optogenetic approach combines high sensitivity and high acuity with superior motion vision, and, unlike optogenetic gene therapy, can be adjusted for dose, upgraded, as new photoswitches are developed, and discontinued at will.

neuroscience↗