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Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane\u2013endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.<\/jats:p>","DOI":"10.1085\/jgp.202012858","type":"journal-article","created":{"date-parts":[[2021,9,4]],"date-time":"2021-09-04T02:20:57Z","timestamp":1630722057000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":12,"title":["EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues"],"prefix":"10.1085","volume":"153","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8499-3449","authenticated-orcid":true,"given":"Parashar","family":"Thapa","sequence":"first","affiliation":[{"name":"Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert","family":"Stewart","sequence":"additional","affiliation":[{"name":"Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2446-3339","authenticated-orcid":true,"given":"Rebecka J.","family":"Sepela","sequence":"additional","affiliation":[{"name":"Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0964-385X","authenticated-orcid":true,"given":"Oscar","family":"Vivas","sequence":"additional","affiliation":[{"name":"Center for Neuroscience, University of California, Davis, Davis, CA 2"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laxmi K.","family":"Parajuli","sequence":"additional","affiliation":[{"name":"Center for Neuroscience, University of California, Davis, Davis, CA 2"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark","family":"Lillya","sequence":"additional","affiliation":[{"name":"Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 1"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sebastian","family":"Fletcher-Taylor","sequence":"additional","affiliation":[{"name":"Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 1"},{"name":"The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 3"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3655-3638","authenticated-orcid":true,"given":"Bruce E.","family":"Cohen","sequence":"additional","affiliation":[{"name":"The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 3"},{"name":"Division of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 4"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karen","family":"Zito","sequence":"additional","affiliation":[{"name":"Center for Neuroscience, University of California, Davis, Davis, CA 2"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6975-982X","authenticated-orcid":true,"given":"Jon T.","family":"Sack","sequence":"additional","affiliation":[{"name":"Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 1"},{"name":"Department of Anesthesiology and Pain Medicine, University of California, Davis, Davis, CA 5"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"291","published-online":{"date-parts":[[2021,9,28]]},"reference":[{"key":"2023072821444134000_bib1","doi-asserted-by":"publisher","first-page":"top70","DOI":"10.1101\/pdb.top70","article-title":"How calcium indicators work","volume":"2010","author":"Adams","year":"2010","journal-title":"Cold Spring Harb. 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