Biology subjects

MacLean, D. M.

Publications and source records attributed to MacLean, D. M..

6 recordsLinked to original sources

bioRxiv2024

The impact of subunit type, alternative splicing, and auxiliary proteins on AMPA receptor trafficking

AMPA receptors underlie fast excitatory synaptic transmission in the mammalian nervous system and are critical for the expression of synaptic plasticity. Four genes encode the AMPA subunits, each subject to RNA editing and alternative splicing at multiple positions. In addition, each tetrameric AMPA receptor can harbor up to four auxiliary proteins, of which there are multiple types. Subunit type, alternative splicing, and auxiliary proteins are all known to affect AMPA receptor gating and trafficking. However, determining which factors dominate AMPA receptor trafficking requires high throughput assessment of trafficking across multiple conditions. Here, we deploy two such methods to assess the relative contribution of AMPA subunit type (GluA1 versus GluA2), alternative splicing (flip versus flop), and various transmembrane AMPA receptor regulatory proteins (TARPs) to AMPA receptor trafficking. We find that subunit type is the most important factor, with GluA2 showing much better surface expression than GluA1, and alternative splicing plays a secondary role, with flip subunits consistently outperforming flop variants in surface expression across all conditions. Type 1 TARPs ({gamma}2-4 and {gamma}8) enhance surface trafficking, while Type 2 TARPs ({gamma}5 and {gamma}7) reduced surface expression, although we could not detect differences within each type. These data will be a helpful resource in comparing surface expression across a variety of AMPA receptor compositions. Our assays will also enable high throughput assessment of novel disease-associated mutations, chimeras, and auxiliary and chaperone proteins.

neuroscience↗

bioRxiv2024

Proline substitutions in the ASIC1 β11-12 linker slow desensitization

Desensitization is a prominent feature of nearly all ligand gated ion channels. Acid-sensing ion channels (ASIC) undergo desensitization within hundreds of milliseconds to seconds upon continual extracellular acidification. The ASIC mechanism of desensitization is primarily due to the isomerization or "flipping" of a short linker joining the 11th and 12th beta sheets in the extracellular domain. In the resting and active states this {beta}11-12 linker adopts an "upward" conformation while in the desensitized conformation the linker assumes a "downward" state. To accommodate this "downward" state, specific peptide bonds within the linker adopt either trans-like or cis-like conformations. Since proline-containing peptide bonds undergo cis-trans isomerization very slowly, we hypothesized that introducing proline residues in the linker may slow or even abolish ASIC desensitization, potentially providing a valuable research tools. Proline substitutions in the chicken ASIC1 {beta}11-12 linker (L414P and Y416P) slowed desensitization decays approximately 100 to 1000-fold as measured in excised patches. Both L414P and Y416P shifted the steady state desensitization curves to more acidic pHs while activation curves and ion selectivity of these slow-desensitizing currents were largely unaffected. To investigate the functional stoichiometry of desensitization in the trimeric ASIC, we created families of L414P and Y416P concatemers with zero, one, two or three proline substitutions in all possible configurations. Introducing one or two L414P or Y416P mutations only slightly attenuated desensitization, suggesting that conformational changes in the remaining faster wild type subunits were sufficient to desensitize the channel. These data highlight the unusual cis-trans isomerization mechanism of ASIC desensitization and support a model where a single subunit is sufficient to desensitize the entire channel.

biophysics↗

bioRxiv2022

PP1 inhibitor-2 promotes PP1γ positive regulation of synaptic transmission

Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1{gamma}, but not PP1, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhances PP1{gamma} interaction with its major synaptic scaffold, neurabin, by Forster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 autoinhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.

neuroscience↗

bioRxiv2021

Topography and motion of the acid-sensing ion channel intracellular domains

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the cASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 [A] and that the N and proximal segment of the C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.

physiology↗

bioRxiv2020

Mutation of a conserved Gln residue does not abolish desensitization of acid-sensing ion channel 1

Desensitization is a common feature of ligand-gated ion channels although the molecular cause varies widely between channel types. Mutations that substantially reduce or abolish desensitization have been described for many ligand-gated ion channels including glutamate, GABA, glycine and nicotinic receptors but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1 the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes essentially like wild type when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, Q276G does not substantially reduce desensitization but surprisingly slows entry to and exit from the desensitized state, thus requiring longer agonist applications to reach equilibrium. Our data reveal that while the Q/G mutation does not substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.

biophysics↗

bioRxiv2019

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

Forster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FPs ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance but are unable to definitively determine the cause, all though protein maturation seems to play a role. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with mScarlet-I out performing mCherry due its higher brightness.

biophysics↗