{
  "paper": "kolb-2026-igabasnfr2",
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    "orphans": [
      {
        "uid": "results-001",
        "section": "results",
        "text": "Results Screening for improved variants The original iGABASnFR1 ( Figure 1a , top) was engineered to increase fluorescence in the presence of GABA and express robustly in mammalian neurons, making it a valuable addition to the suite of biosensors available to study the brain ( Dong et al., 2022 ; Looger and Griesbeck, 2012 ; Marvin et al., 2019 ; Yang et al., 2024 ).",
        "stats": [],
        "panels": [
          "fig1a"
        ]
      },
      {
        "uid": "results-009",
        "section": "results",
        "text": "Numbering is relative to each of the constituent protein domains; the relationship to sequential numbering of the entire polypeptide is shown in Figure 1\u2014figure supplement 1 .",
        "stats": [],
        "panels": [
          "fig1s1"
        ]
      },
      {
        "uid": "results-023",
        "section": "results",
        "text": "Figure 1\u2014figure supplement 1. Annotated amino acid sequence of iGABASnFRs.",
        "stats": [],
        "panels": [
          "fig1s1"
        ]
      },
      {
        "uid": "results-037",
        "section": "results",
        "text": "Specifically, we identified 39 sites to target: 14 near the GABA binding site, 6 at or around the protein hinge area, 10 on the cpSFGFP, and 9 at the interface between the two ligand-binding and cpGFP domains ( Figure 1a , bottom).",
        "stats": [],
        "panels": [
          "fig1a"
        ]
      },
      {
        "uid": "results-059",
        "section": "results",
        "text": "For 10 action potentials (APs), iGABASnFR2 exhibited a peak \u0394F/F 4.1-fold greater than iGABASnFR1 (iGABASnFR2 0.77\u00b10.13; iGABASnFR1 0.19\u00b10.02; p <0.001 Tukey\u2019s HSD post hoc test following one-way ANOVA), and a signal-to-noise ratio (SNR) threefold higher (iGABASnFR2 67.6\u00b111.9; iGABASnFR1 22.7\u00b15.5; p <0.001).",
        "stats": [
          "p <0.001",
          "p <0.001"
        ],
        "panels": []
      },
      {
        "uid": "results-060",
        "section": "results",
        "text": "Rise time constants were slightly faster (iGABASnFR2 43\u00b19 ms; iGABASnFR1 61\u00b113 ms; p <0.001), while decay times were slower (iGABASnFR2 73\u00b126 ms; iGABASnFR1 62\u00b129 ms; p <0.001).",
        "stats": [
          "p <0.001",
          "p <0.001"
        ],
        "panels": []
      },
      {
        "uid": "results-061",
        "section": "results",
        "text": "The negative-going iGABASnFR2n exhibited slightly reduced performance relative to iGABASnFR2 but still achieved a 3.3-fold greater peak \u0394F/F (iGABASnFR2n 0.62\u00b10.12; iGABASnFR1 0.19\u00b10.02; p <0.001), and 40% higher SNR compared to iGABASnFR1 (iGABASnFR2n 32.5\u00b17.5; iGABASnFR1 22.7\u00b15.5; p <0.01).",
        "stats": [
          "p <0.001",
          "p <0.01"
        ],
        "panels": []
      },
      {
        "uid": "results-066",
        "section": "results",
        "text": "Traces and error bars denote mean \u00b1 s.e.m., n=20 culture wells for each variant.",
        "stats": [
          "n=20"
        ],
        "panels": []
      },
      {
        "uid": "results-067",
        "section": "results",
        "text": "( c ) Rise time constants of the three sensor variants obtained from exponential fits for the 1 AP condition (n=24 culture wells for iGABASnFR1 and iGABASnFR2, n=18 for iGABASnFR2n).",
        "stats": [
          "n=24",
          "n=18"
        ],
        "panels": []
      },
      {
        "uid": "results-084",
        "section": "results",
        "text": "Within that hinge region, the F101L mutation, introduced in the original iGABASnFR1 ( Marvin et al., 2019 ), increased binding affinity 10-fold ( Figure 3b ).",
        "stats": [],
        "panels": [
          "fig3b"
        ]
      },
      {
        "uid": "results-086",
        "section": "results",
        "text": "The GABA binding site itself is defined by the side chains of W9, T13, F100, Y102, W202, R205, D228, and Y264 ( Figure 3c ).",
        "stats": [],
        "panels": [
          "fig3c"
        ]
      },
      {
        "uid": "results-091",
        "section": "results",
        "text": "In iGABASnFR2, the L178 gfp S mutation at this interface is likely involved in a hydrogen-bonding network with nearby hydrophilic residues, enhancing the GABA-induced fluorescence change ( Figure 3b ).",
        "stats": [],
        "panels": [
          "fig3b"
        ]
      },
      {
        "uid": "results-103",
        "section": "results",
        "text": "Somewhat surprisingly, purified iGABASnFR2 showed a smaller dynamic range than iGABASnFR1, although its affinity was higher ( Figure 4a ; max dF/F0 iGABASnFR2: 0.45, iGABASnFR1: 1.82; EC 50 iGABASnFR2=1.1 \u03bcM, iGABASnFR1=5.7 \u03bcM).",
        "stats": [],
        "panels": [
          "fig4a"
        ]
      },
      {
        "uid": "results-113",
        "section": "results",
        "text": "Lines indicate fits to mean of n=5 titration series, error bars are s.e.m.",
        "stats": [
          "n=5"
        ],
        "panels": []
      },
      {
        "uid": "results-122",
        "section": "results",
        "text": "Lines indicate fits to mean of n=3 replicates from three separate batches of purified protein.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "results-128",
        "section": "results",
        "text": "Each titration has a minimum of n=3 replicates.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "results-130",
        "section": "results",
        "text": "Different iGABASnFR variants (200 nM) were titrated with increasing concentrations of GABA in the presence of different potential competing compounds at 1 mM. Lines indicate fits to mean of n=3 titration series, error bars are s.e.m.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "results-136",
        "section": "results",
        "text": "Data points represent the mean of n=3 technical replicates; error bars indicate s.e.m.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "results-144",
        "section": "results",
        "text": "Additional biophysical properties are reported in Table 1 .",
        "stats": [],
        "panels": [
          "table1"
        ]
      },
      {
        "uid": "results-147",
        "section": "results",
        "text": "Table 1. Photophysical properties of iGABASnFR variants as purified proteins. \u03bb abs (nm) \u03bb Ex (nm) \u03bb Em (nm) \u0394F/F \u03b5 (M/cm 2 ) \u03a6 \u03c4 (ns) GABA PBS GABA PBS GABA PBS iGABASnFR1 490 489 508 1.9 14,070\u00b1600 5375\u00b1220 0.58\u00b10.01 0.54\u00b10.03 2.22\u00b10.05 2.32\u00b10.01 iGABASnFR2 490 490 508 0.46 26,195\u00b1390 19,800\u00b1380 0.61\u00b10.02 0.54\u00b10.01 2.29\u00b10.08 2.30\u00b10.02 iGABASnFR2n 493 496 509 \u20130.13 12,390\u00b1690 13,000\u00b1220 0.72\u00b10.02 0.76\u00b10.01 2.71\u00b10.02 2.72\u00b10.02 Evaluating sensor performance in intact retina To evaluate indicator performance in an intact neural circuit, we examined synaptic transmission in the retina.",
        "stats": [],
        "panels": [
          "table1"
        ]
      },
      {
        "uid": "results-152",
        "section": "results",
        "text": "Specifically, the hypothesis is that GABA release is stronger when a visual stimulus moves so that excitation sweeps down the dendritic branch from root to tip (centrifugal motion) rather than when it sweeps from tip to root (centripetal motion) ( Figure 5a ).",
        "stats": [],
        "panels": [
          "fig5a"
        ]
      },
      {
        "uid": "results-153",
        "section": "results",
        "text": "The resulting direction-selective inhibition is relayed to direction-selective retinal ganglion cells (DSGCs) through asymmetric synaptic connections ( Briggman et al., 2011 ; Yonehara et al., 2011 ; Figure 5b ).",
        "stats": [],
        "panels": [
          "fig5b"
        ]
      },
      {
        "uid": "results-160",
        "section": "results",
        "text": "( f\u2013h ) Results of imaging using iGABASnFR2. ( i ) Histograms of response amplitude index (left) and response reliability (right) from SACs expressing iGABASnFR. n=147 ROIs collected across five retinae.",
        "stats": [
          "n=147"
        ],
        "panels": []
      },
      {
        "uid": "results-161",
        "section": "results",
        "text": "( j ) As in i but with SACs expressing iGABASnFR2. n=346 ROIs collected from three retinae.",
        "stats": [
          "n=346"
        ],
        "panels": []
      },
      {
        "uid": "results-164",
        "section": "results",
        "text": "Line and shading indicate mean \u00b1 s.d (n=147 for iGABASnFR, n=346 for iGABASnFR2).",
        "stats": [
          "n=147",
          "n=346"
        ],
        "panels": []
      },
      {
        "uid": "results-165",
        "section": "results",
        "text": "( l ) Comparison of signal-to-noise ratio (SNR) of the motion response detected with the two sensor versions. p =0. Two-tailed Mann-Whitney-Wilcoxon test.",
        "stats": [
          "p =0"
        ],
        "panels": []
      },
      {
        "uid": "results-166",
        "section": "results",
        "text": "( m ) Comparison of direction selectivity (CV, circular variance) for the two sensor versions. p =7.812\u00d710 \u20136 .",
        "stats": [
          "p =7.812"
        ],
        "panels": []
      },
      {
        "uid": "captions-004",
        "section": "captions",
        "text": "Numbering is relative to each of the constituent protein domains; the relationship to sequential numbering of the entire polypeptide is shown in Figure 1\u2014figure supplement 1 .",
        "stats": [],
        "panels": [
          "fig1s1"
        ]
      },
      {
        "uid": "captions-018",
        "section": "captions",
        "text": "[panels detected: a, b, c, d] === Figure 1s1 === Figure 1\u2014figure supplement 1. Annotated amino acid sequence of iGABASnFRs.",
        "stats": [],
        "panels": [
          "fig1s1"
        ]
      },
      {
        "uid": "captions-029",
        "section": "captions",
        "text": "Traces and error bars denote mean \u00b1 s.e.m., n=20 culture wells for each variant.",
        "stats": [
          "n=20"
        ],
        "panels": []
      },
      {
        "uid": "captions-030",
        "section": "captions",
        "text": "( c ) Rise time constants of the three sensor variants obtained from exponential fits for the 1 AP condition (n=24 culture wells for iGABASnFR1 and iGABASnFR2, n=18 for iGABASnFR2n).",
        "stats": [
          "n=24",
          "n=18"
        ],
        "panels": []
      },
      {
        "uid": "captions-044",
        "section": "captions",
        "text": "Lines indicate fits to mean of n=5 titration series, error bars are s.e.m.",
        "stats": [
          "n=5"
        ],
        "panels": []
      },
      {
        "uid": "captions-053",
        "section": "captions",
        "text": "Lines indicate fits to mean of n=3 replicates from three separate batches of purified protein.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "captions-060",
        "section": "captions",
        "text": "Different iGABASnFR variants (200 nM) were titrated with increasing concentrations of GABA in the presence of different potential competing compounds at 1 mM. Lines indicate fits to mean of n=3 titration series, error bars are s.e.m.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "captions-065",
        "section": "captions",
        "text": "Data points represent the mean of n=3 technical replicates; error bars indicate s.e.m.",
        "stats": [
          "n=3"
        ],
        "panels": []
      },
      {
        "uid": "captions-072",
        "section": "captions",
        "text": "( f\u2013h ) Results of imaging using iGABASnFR2. ( i ) Histograms of response amplitude index (left) and response reliability (right) from SACs expressing iGABASnFR. n=147 ROIs collected across five retinae.",
        "stats": [
          "n=147"
        ],
        "panels": []
      },
      {
        "uid": "captions-073",
        "section": "captions",
        "text": "( j ) As in i but with SACs expressing iGABASnFR2. n=346 ROIs collected from three retinae.",
        "stats": [
          "n=346"
        ],
        "panels": []
      },
      {
        "uid": "captions-076",
        "section": "captions",
        "text": "Line and shading indicate mean \u00b1 s.d (n=147 for iGABASnFR, n=346 for iGABASnFR2).",
        "stats": [
          "n=147",
          "n=346"
        ],
        "panels": []
      },
      {
        "uid": "captions-077",
        "section": "captions",
        "text": "( l ) Comparison of signal-to-noise ratio (SNR) of the motion response detected with the two sensor versions. p =0. Two-tailed Mann-Whitney-Wilcoxon test.",
        "stats": [
          "p =0"
        ],
        "panels": []
      },
      {
        "uid": "captions-078",
        "section": "captions",
        "text": "( m ) Comparison of direction selectivity (CV, circular variance) for the two sensor versions. p =7.812\u00d710 \u20136 .",
        "stats": [
          "p =7.812"
        ],
        "panels": []
      },
      {
        "uid": "tables-001",
        "section": "tables",
        "text": "Table 1. Photophysical properties of iGABASnFR variants as purified proteins. \u03bb abs (nm) \u03bb Ex (nm) \u03bb Em (nm) \u0394F/F \u03b5 (M/cm 2 ) \u03a6 \u03c4 (ns) GABA PBS GABA PBS GABA PBS iGABASnFR1 490 489 508 1.9 14,070\u00b1600 5375\u00b1220 0.58\u00b10.01 0.54\u00b10.03 2.22\u00b10.05 2.32\u00b10.01 iGABASnFR2 490 490 508 0.46 26,195\u00b1390 19,800\u00b1380 0.61\u00b10.02 0.54\u00b10.01 2.29\u00b10.08 2.30\u00b10.02 iGABASnFR2n 493 496 509 \u20130.13 12,390\u00b1690 13,000\u00b1220 0.72\u00b10.02 0.76\u00b10.01 2.71\u00b10.02 2.72\u00b10.02",
        "stats": [],
        "panels": [
          "table1"
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}