Plain wording

The fluorescence attributed to iGABASnFR2 comes from compounds that resemble GABA rather than from GABA itself.

slug alt-signal-from-cross-reactivityrole hypothesis

The crystal structure of iGABASnFR2, showing its binding pocket and cpGFP arrangement, is deposited in the Protein Data Bank.

slug crystal-structure-pdb-9d57role methodologicalpanel fig3 (or structural supplement)

A sufficiently sensitive GABA sensor enables measurements iGABASnFR1 cannot make at all, rather than cleaner versions of old ones.

slug hypothesis-improved-sensor-enables-new-biologyrole hypothesispanel hypothesis

Mutating a handful of residues around iGABASnFR1's GABA-binding pocket and cpGFP linkers yields a far better sensor without losing selectivity.

slug hypothesis-saturation-mutagenesis-yields-improved-sensorrole hypothesispanel hypothesis

iGABASnFR2 produces about thirteen times as many responding pixels as iGABASnFR1, reflecting better sensitivity and better membrane trafficking.

slug igabasnfr2-13fold-expression-increaserole empiricalpanel fig1C

iGABASnFR2 works under two-photon imaging, and both new variants are less sensitive to pH than iGABASnFR1.

slug igabasnfr2-2p-compatiblerole methodologicalpanel fig4e, fig4f, fig4-supplement3

GABA binding closes iGABASnFR2's binding lobes but leaves the fluorescent cpGFP domain almost unmoved, unlike GCaMP with calcium.

slug igabasnfr2-cpgfp-rigid-on-gaba-bindingrole interpretationpanel fig3a

iGABASnFR2 gives a roughly fourfold larger fluorescence change than iGABASnFR1 for the same stimulation in cultured neurons.

slug igabasnfr2-fourfold-sensitivity-gainrole empiricalpanel fig1, fig2

iGABASnFR2 responds to GABA and not to structurally similar compounds, none of which block or distort its GABA response.

slug igabasnfr2-gaba-selective-specificityrole controlpanel fig4-supplement1, fig4-supplement2

iGABASnFR2 detects GABA spilling into the extracellular space of mouse barrel cortex when the whiskers are stimulated.

slug igabasnfr2-invivo-barrel-cortexrole empiricalpanel fig6c, fig6-video1

iGABASnFR2 rises faster and decays more slowly than iGABASnFR1, while the negative-going variant rises more slowly than both.

slug igabasnfr2-kinetics-rise-decayrole empiricalpanel fig2c, fig2d

On the neuron surface iGABASnFR2 binds GABA about seven times more tightly than iGABASnFR1, still above resting GABA levels.

slug igabasnfr2-oncell-affinity-sevenfoldrole empiricalpanel fig4b

iGABASnFR2 shows that starburst amacrine cells release GABA differently depending on motion direction, which iGABASnFR1 could not resolve.

slug igabasnfr2-retina-direction-selectivityrole empiricalpanel fig5

iGABASnFR2 detects GABA released from single hippocampal interneuron boutons, where iGABASnFR1 gave no signal at all.

slug igabasnfr2-single-bouton-hippocampusrole empiricalpanel fig6a, fig6b

iGABASnFR2 and its negative-going variant bind GABA in one fast step, while iGABASnFR1 binds in two slower phases.

slug igabasnfr2-single-exponential-kineticsrole empiricalpanel fig4c, fig4d

A variant called iGABASnFR2n dims rather than brightens when GABA binds, and is also far better expressed than iGABASnFR1.

slug igabasnfr2n-negative-going-variantrole empiricalpanel fig1C, fig1D

A screen of nearly 4,000 variants at 39 sites yielded 93 more responsive than iGABASnFR1 and 22 better in both response and expression.

slug mutagenesis-3947-variants-screenedrole methodologicalpanel fig1B, fig1C

iGABASnFR2 should beat iGABASnFR1 at single boutons, at single-trial direction selectivity in retina, and at whisker-evoked GABA in the living brain.

slug prediction-improved-sensor-enables-new-measurementsrole predictionpanel prediction

The screen should yield several variants beating iGABASnFR1 on response, at least one also better expressed, and some behaving in new ways.

slug prediction-screen-yields-multiple-improved-variantsrole predictionpanel prediction

All tests use cultured neurons, purified protein, retina, hippocampal slice and mouse cortex, not awake or non-mammalian preparations.

slug scope-sensor-engineering-paperrole scopepanel scope

The performance claims rest on bench measurements that cannot be reproduced from the deposited code and data, which only redraw the figures.

slug screening-scope-wet-lab-onlyrole scopepanel all figures (assessment)