Detection of changes in near-membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator.
A Ca2+ indicator has been synthesized and characterized which can be used to monitor rapid changes in the free Ca2+ concentration ([Ca2+]) immediately adjacent to cell membranes. This indicator, referred to as C18-Fura-2, consists of a Fura-2 molecule conjugated to a lipophilic alkyl chain which will insert into cell membranes. When associated with cell membranes in low concentrations, C18-Fura-2 exhibits an excitation spectrum with a large Stokes shift and a single isobestic point, thus [Ca2+] can be calculated ratiometrically. The apparent Ca2+ dissociation constant of cell-associated C18-Fura-2 is around 150 nM. C18-Fura-2 orients in the cell membrane so that the fluorophore is facing the side to which it was applied. C18-Fura-2 was used to record rapid changes in intracellular [Ca2+] which occurred in response to membrane depolarization in isolated smooth muscle cells. The initial rise of the [Ca2+] transient reported by C18-Fura-2 was four to six times faster than the rise of the [Ca2+] transient reported by cytosolic Fura-2. This result suggests that C18-Fura-2 was located at the plasma membrane near sites of Ca2+ influx and indicates that membrane-associated Ca2+ indicators can be used to detect rapid, localized changes in [Ca2+] which are obscured in signals recorded using water-soluble, bulk cytosolic fluorescent Ca2+ indicators.
Etter
E F
EF
Department of Physiology, University of Massachusetts Medical School, Worcester 01605.
Kuhn
M A
MA
Fay
F S
FS
D000818:Animals; D002024:Bufo marinus; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D005753:Gastric Mucosa / Q000378:metabolism; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence / Q000379:methods; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013270:Stomach / Q000166:cytology
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C086614:C18-fura-2; D002118:Calcium; D016257:Fura-2