John Dumas | PubMed Health

PubMed2004

Cardiac optical mapping under a translucent stimulation electrode.

Major effects of stimulation on cardiac transmembrane potentials (Vm) are thought to occur under the electrode, however these have not been optically mapped due to blockage of light by electrodes. Here we optically mapped under translucent indium tin oxide (ITO) electrodes in hearts stained with transmembrane voltage sensitive fluorescent dye, di-4-ANEPPS excited at 488 nm. Emissions in wavelength bands 510-570 nm and >590 nm were similarly affected by changes in ITO transmittance due to electrochemical effects of current at the electrode interface. Dual-wavelength ratiometric mapping with the two emission bands revealed Vm under the electrode during plateau-phase stimulation (220 mA). Changes in Vm were heterogeneous under the electrode, and were anisotropic with larger values along the fiber axis. These results explain early excitation sites for sufficiently strong diastolic stimulation, and agree with theoretical predictions based on summation of anisotropic effects of point stimulation and a linear 3-d cardiac bidomain computer model. The bidomain model and experiments disagree under the edge of the electrode, where modeled Vm is much larger. Thus, changes in Vm under an electrode are anisotropic with greater Vm in the direction parallel to fibers. Nonlinear effects of stimulation in hearts may limit changes in Vm under the electrode edge.

Animals↗

PubMed2001

Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1.

Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.

Amino Acid Sequence↗