LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Kenneth Dakin, YuRui Zhao, Wen Hong Li

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.

Original languageEnglish (US)
Pages (from-to)55-62
Number of pages8
JournalNature Methods
Volume2
Issue number1
DOIs
StatePublished - Jan 2005

Fingerprint

Gap Junctions
Assays
Coloring Agents
Imaging techniques
Communication
Ionomycin
Connexins
Fluorophores
Fluorescence microscopy
Cell membranes
Fluorescent Dyes
Labels
Chemical activation
Cells
Molecular Probes
Cell Size
Fluorescence Microscopy
Permeability
Cell Count
Cell Membrane

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Cite this

LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling. / Dakin, Kenneth; Zhao, YuRui; Li, Wen Hong.

In: Nature Methods, Vol. 2, No. 1, 01.2005, p. 55-62.

Research output: Contribution to journalArticle

@article{b8eca2ddd21b4ad187015931279516cb,
title = "LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling",
abstract = "Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.",
author = "Kenneth Dakin and YuRui Zhao and Li, {Wen Hong}",
year = "2005",
month = "1",
doi = "10.1038/nmeth730",
language = "English (US)",
volume = "2",
pages = "55--62",
journal = "Nature Methods",
issn = "1548-7091",
publisher = "Public Library of Science",
number = "1",

}

TY - JOUR

T1 - LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

AU - Dakin, Kenneth

AU - Zhao, YuRui

AU - Li, Wen Hong

PY - 2005/1

Y1 - 2005/1

N2 - Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.

AB - Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.

UR - http://www.scopus.com/inward/record.url?scp=18744395238&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=18744395238&partnerID=8YFLogxK

U2 - 10.1038/nmeth730

DO - 10.1038/nmeth730

M3 - Article

C2 - 15782161

AN - SCOPUS:18744395238

VL - 2

SP - 55

EP - 62

JO - Nature Methods

JF - Nature Methods

SN - 1548-7091

IS - 1

ER -