Biology subjects

Plested, A. J. R.

Publications and source records attributed to Plested, A. J. R..

6 recordsLinked to original sources

bioRxiv2022

State-dependent photocrosslinking unveiled the role of intersubunit interfaces on Ca2+ activating mechanisms of the BK channel

BK channels are high-conductance potassium channels that are activated by voltage and Ca2+. The pore-forming -subunits form homotetramers including a membrane-spanning domain and a cytosolic domain with a tandem of RCK-like domains (RCK1 and RCK2) per subunit. The eight RCKs compose high affinity Ca2+-binding sites that drive channel activation. Full-length Cryo-EM structures revealed intersubunit interactions between the RCK domains. Asparagine 449 (N449, human BK channel) is located at the RCK1 domain and coordinates Ca2+ in the RCK2 of the adjacent subunit. In addition, two Arginines (R786 and R790) of one RCK2 and Glutamate 955 of the adjacent subunit constitute an additional interaction interface. Functional studies on these residues showed that these two interfaces are crucial in Ca2+ sensitivity. To detect structural rearrangements induced by Ca2+ during channel activation, we took advantage of the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine (BzF). Functional channels were obtained with this amino acid inserted at 11 positions. N449 and R786 positions (N449BzF and R786BzF respectively). UV-induced photocrosslinking led to Ca2+ dependent and voltage-independent effects in both mutants. N449BzF showed a steady-state current reduction at saturating Ca2+ concentrations. Our data shows that this effect mainly relies on full occupancy of the RCK1 Ca2+ binding site, since mutation of this site abolished the effect. The R786BzF construct showed a substantial potentiation of the current in the absence of Ca2+. In this case, photocrosslinking seems to favor the activation of the channel by voltage. Overall, these results suggest mobile interfaces between RCK domains are key to BK channel activation.

biophysics↗

bioRxiv2022

Asymmetry and ion selectivity properties of bacterial channel NaK mutants mimicking ionotropic glutamate receptors

Ionotropic glutamate receptors are ligand-gated cation channels that play essential roles in the excitatory synaptic transmission throughout the central nervous system. A number of open-pore structures of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid (AMPA)-type glutamate receptors became recently available by cryo-electron microscopy (cryo-EM). These structures provide valuable insights into the conformation of the selectivity filter (SF), the part of the ion channel that determines the ion selectivity. Nonetheless, due to the moderate resolution of the cryo-EM structures, detailed information such as ion occupancy of monovalent and divalent cations as well as exact displacement of the side-chains in the SF is still missing. Here, in order to resolve high-resolution crystal structures of the AMPA SF in its open-state, we incorporated the partial SF sequence of the AMPA receptor into the bacterial tetrameric cation channel NaK, which served as a structural scaffold. We determined a series of X-ray structures of NaK-CDI, NaK-SDI and NaK-SELM mutants at 1.42-2.10 [A] resolution, showing distinct ion occupation of different monovalent cations. Molecular dynamics (MD) simulations of NaK-CDI indicated the channel to be conductive to monovalent cations, which agrees well with our electrophysiology recordings. Moreover, unique structural asymmetry of the SF was revealed by the X-ray structures and MD simulations, implying its importance in ion non-selectivity of tetrameric cation channels.

biophysics↗

bioRxiv2022

CaMKII activity spreads by inter-holoenzyme phosphorylation

The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.

biophysics↗

bioRxiv2021

Targeted sensors for glutamatergic neurotransmission

Optical report of neurotransmitter release allows visualization of excitatory synaptic transmission. Sensitive genetically-encoded glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-{gamma}2 and SnFR-{gamma}8, retained function and reported spontaneous glutamate release in hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic neurons on micro island cultures, evoked release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-{gamma}2 is a linear reporter of the global quantal parameters and short term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-{gamma}2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

neuroscience↗

bioRxiv2020

Non-selective cation permeation in an AMPA-type glutamate receptor

Fast excitatory synaptic transmission in the central nervous system relies on the AMPA-type glutamate receptor (AMPAR). This receptor incorporates a non-selective cation channel which is opened by the binding of glutamate. Although the open pore structure has recently became available from cryo-electron microscopy (Cryo-EM), the molecular mechanisms governing cation permeability in AMPA receptors are not understood. Here, we combined microsecond molecular dynamics (MD) simulations on a putative open state structure of GluA2 with electrophysiology on cloned channels to elucidate ion permeation mechanisms. Na+, K+ and Cs+ permeated at physiological rates, consistent with a structure that represents a true open state. A single major ion binding site for Na+ and K+ in the pore represents the simplest selectivity filter (SF) structure for any tetrameric cation channel of known structure. The minimal SF comprised only Q586 and Q587, and other residues on the cytoplasmic side formed a cone- shaped void that lacked major interactions with ions. We observed Cl- invasion of the upper pore, explaining anion permeation in the edited form of GluA2. A permissive architecture of the SF accommodated different alkali metals in distinct solvation states to allow rapid, non-selective cation permeation, and co-permeation by water. Simulations suggested Cs+ uses two equally populated ion binding sites in the filter and we confirmed with electrophysiology of GluA2 that Cs+ is more permeant than Na+, consistent with serial binding sites preferentially driving selectivity. Significance StatementAMPA-type glutamate receptors (AMPARs) are key actors in neurotransmission, making the final step in a relay of excitability from one brain cell to another. The receptor contains an integral ion channel, which, when opened by neurotransmitter binding, permits sodium and other cations to cross the cell membrane. We investigated permeation of sodium, potassium and caesium in an AMPAR at the atomistic level using a computational molecular dynamics approach on a structure with the ion channel pore in a presumably open state. We determined that the region selecting between cations is the simplest of any channel of this type. Distinct from ion channels that select single ion species, cations are never fully dehydrated and have only one major ion binding site in the filter. Simulations suggested two similar binding sites for caesium, and studies of AMPARs in mammalian cell membranes showed that this makes caesium more permeant than sodium.

biophysics↗

bioRxiv2018

Auxiliary subunits keep AMPA receptors compact during activation and desensitization

Signal transduction at vertebrate excitatory synapses involves the activity of ionotropic glutamate receptors, including the AMPA (-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor. Technical advances in cryo-electron microscopy have brought a slew of full-length structures of AMPA receptors, on their own and in combination with auxiliary subunits. These structures illustrate a wide range of conformations, indicating that individual domains might undergo substantial lateral motions during gating, resulting in an open, \"relaxed\" extracellular layer. Here, we used bifunctional methanethiosulfonate cross-linkers to calibrate the conformations found in functional AMPA receptors both in the presence and absence of the auxiliary subunit Stargazin. Our data indicate that AMPA receptors have considerable conformational freedom and can get trapped in stable, relaxed conformations, especially upon long exposures to glutamate. In contrast, Stargazin limits this conformational flexibility. Thus, under synaptic conditions, where brief glutamate exposures and the presence of Stargazin dominate, AMPA receptors are unlikely to adopt very relaxed conformations during gating.

biophysics↗