Single channel study of the spasmodic mutation α1A52S in recombinant rat glycine receptors

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic, that has a startle phenotype, has a point mutation (A52S) in the glycine receptor α1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell-attached patch clamp recording of single-channel currents elicited by 30 – 10000 μM glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wildtype receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the ‘flip’ mechanism, suggests that pre-opening isomerisation to the flipped conformation that follows binding is less favoured in mutant than in wild-type receptors, and, especially, that the flipped conformation has a 100-fold lower affinity for glycine than in wildtype receptors. In contrast, the efficacy of the gating reaction was similar to that of wild-type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC50 for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H(+), Na(+), K(+) and anions have been discovered or engineered, Ca(+)-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca(+) permeability and improve their Ca(+) affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca(+) permeation at negative voltage and low extracellular Ca(+) concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca(+) concentrations. Moreover, CapChR2 can robustly trigger Ca(+) signalling in hippocampal neurons. When expressed together with genetically encoded Ca(+) indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca(+) entry in brain explants

Selective endocytosis of Ca(2+)-permeable AMPARs by the Alzheimer's disease risk factor CALM bidirectionally controls synaptic plasticity

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca(2+)-permeable (CP) versus heteromeric Ca(2+)-impermeable (CI) AMPARs. Here, we report that the Alzheimer's disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain-containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain

Mechanism of Calcium Permeation in a Glutamate Receptor Ion Channel

The amp; 945; amino 3 hydroxy 5 methyl 4 isoxazolepropionic acid receptors AMPARs are neurotransmitter activated cation channels ubiquitously expressed in vertebrate brains. The regulation of calcium flux through the channel pore by RNA editing is linked to synaptic plasticity while excessive calcium influx poses a risk for neurodegeneration. Unfortunately, the molecular mechanisms underlying this key process are mostly unknown. Here, we investigated calcium conduction in calcium permeable AMPAR using Molecular Dynamics MD simulations with recently introduced multisite force field parameters for Ca2 . Our calculations are consistent with experiment and explain the distinct calcium permeability in different RNA edited forms of GluA2. For one of the identified metal binding sites, multiscale Quantum Mechanics Molecular Mechanics QM MM simulations further validated the results from MD and revealed small but reproducible charge transfer between the metal ion and its first solvation shell. In addition, the ion occupancy derived from MD simulations independently reproduced the Ca2 binding profile in an X ray structure of an NaK channel mimicking the AMPAR selectivity filter. This integrated study comprising X ray crystallography, multisite MD, and multiscale QM MM simulations provides unprecedented insights into Ca2 permeation mechanisms in AMPARs, and paves the way for studying other biological processes in which Ca2 plays a pivotal rol

Dynamics of the ligand binding domain layer during AMPA receptor activation

Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample

CaMKII autophosphorylation can occur between holoenzymes without subunit exchange

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 kinas