Fast GCaMPs for improved tracking of neuronal activity

Xiaonan R. Sun; Aleksandra Badura; Diego A. Pacheco; Laura A. Lynch; Eve R. Schneider; Matthew P. Taylor; Ian B. Hogue; Lynn W. Enquist; Mala Murthy; Samuel S.H. Wang

doi:10.1038/ncomms3170

Fast GCaMPs for improved tracking of neuronal activity

Xiaonan R. Sun, Aleksandra Badura, Diego A. Pacheco, Laura A. Lynch, Eve R. Schneider, Matthew P. Taylor, Ian B. Hogue, Lynn W. Enquist, Mala Murthy, Samuel S.H. Wang

Research output: Contribution to journal › Article › peer-review

105 Scopus citations

Abstract

The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca²⁺-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca²⁺-chelating residues. Second, we find that off-responses to Ca²⁺ are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca²⁺ and by slow binding to the C-lobe at lower Ca²⁺. We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of K_D, allowing coexpression of multiple variants to span an expanded range of Ca²⁺ concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.

Original language	English (US)
Article number	2170
Journal	Nature communications
Volume	4
DOIs	https://doi.org/10.1038/ncomms3170
State	Published - Jul 18 2013
Externally published	Yes

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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@article{2dcce2a443614ca9b17a1040773cc4ee,

title = "Fast GCaMPs for improved tracking of neuronal activity",

abstract = "The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca2+-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca2+-chelating residues. Second, we find that off-responses to Ca2+ are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca2+ and by slow binding to the C-lobe at lower Ca2+. We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of KD, allowing coexpression of multiple variants to span an expanded range of Ca2+ concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.",

author = "Sun, {Xiaonan R.} and Aleksandra Badura and Pacheco, {Diego A.} and Lynch, {Laura A.} and Schneider, {Eve R.} and Taylor, {Matthew P.} and Hogue, {Ian B.} and Enquist, {Lynn W.} and Mala Murthy and Wang, {Samuel S.H.}",

note = "Funding Information: This work was supported by NIH R01 NS045193, (S.S.-H.W.) RC1 NS068414 (L.W.E./ S.S.-H.W.), and P40 RR18604 and NS060699 (L.W.E.), a McKnight Technological Innovations Award (S.S.-H.W.), a W.M. Keck Foundation Distinguished Young Investigator award (S.S.-H.W.), an Alfred P. Sloan Research Fellowship, Klingenstein, McKnight, and NSF CAREER Young Investigator awards (M.M.), and an American Cancer Society Postdoctoral Research Fellowship (M.P.T./I.B.H.). We thank Smita Patel for advice and equipment access for stopped-flow fluorimetry, Fred Hughson for plasmids and protein purification materials, Loren Looger and Jasper Akerboom for advice and GCaMP constructs, Timothy Tayler for assistance with establishing Drosophila lines, Fred Hughson for advice and the gift of BL21(DE3) E. coli, and Steve Lin, Daniel Chang, Tamar Friling, and Yulia Lampi for assistance in experiments. Publisher Copyright: {\textcopyright} 2013 Macmillan Publishers Limited. All rights reserved.",

year = "2013",

month = jul,

day = "18",

doi = "10.1038/ncomms3170",

language = "English (US)",

volume = "4",

journal = "Nature communications",

issn = "2041-1723",

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TY - JOUR

T1 - Fast GCaMPs for improved tracking of neuronal activity

AU - Sun, Xiaonan R.

AU - Badura, Aleksandra

AU - Pacheco, Diego A.

AU - Lynch, Laura A.

AU - Schneider, Eve R.

AU - Taylor, Matthew P.

AU - Hogue, Ian B.

AU - Enquist, Lynn W.

AU - Murthy, Mala

AU - Wang, Samuel S.H.

N1 - Funding Information: This work was supported by NIH R01 NS045193, (S.S.-H.W.) RC1 NS068414 (L.W.E./ S.S.-H.W.), and P40 RR18604 and NS060699 (L.W.E.), a McKnight Technological Innovations Award (S.S.-H.W.), a W.M. Keck Foundation Distinguished Young Investigator award (S.S.-H.W.), an Alfred P. Sloan Research Fellowship, Klingenstein, McKnight, and NSF CAREER Young Investigator awards (M.M.), and an American Cancer Society Postdoctoral Research Fellowship (M.P.T./I.B.H.). We thank Smita Patel for advice and equipment access for stopped-flow fluorimetry, Fred Hughson for plasmids and protein purification materials, Loren Looger and Jasper Akerboom for advice and GCaMP constructs, Timothy Tayler for assistance with establishing Drosophila lines, Fred Hughson for advice and the gift of BL21(DE3) E. coli, and Steve Lin, Daniel Chang, Tamar Friling, and Yulia Lampi for assistance in experiments. Publisher Copyright: © 2013 Macmillan Publishers Limited. All rights reserved.

PY - 2013/7/18

Y1 - 2013/7/18

N2 - The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca2+-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca2+-chelating residues. Second, we find that off-responses to Ca2+ are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca2+ and by slow binding to the C-lobe at lower Ca2+. We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of KD, allowing coexpression of multiple variants to span an expanded range of Ca2+ concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.

AB - The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca2+-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca2+-chelating residues. Second, we find that off-responses to Ca2+ are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca2+ and by slow binding to the C-lobe at lower Ca2+. We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of KD, allowing coexpression of multiple variants to span an expanded range of Ca2+ concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.

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U2 - 10.1038/ncomms3170

DO - 10.1038/ncomms3170

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SN - 2041-1723

VL - 4

JO - Nature communications

JF - Nature communications

M1 - 2170

ER -

Fast GCaMPs for improved tracking of neuronal activity

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