Optimization of a mammalian expression system for the measurement
of sodium channel gating currents.
Sheets, Michael F., John W. Kyle, Stephanie Krueger, and Dorothy A.
Hanck.
The Department of Medicine & The Feinberg Cardiovascular
Research Institute, Northwestern University Medical School and The
Departments of Medicine & Pharmacological and Physiological
Sciences, University of Chicago, Chicago, Illinois
APStracts 3:0161C, 1996.
We describe a new mammalian expression system that optimizes
conditions for the measurement of sodium channel gating currents
(Ig). The small magnitude of gating currents limits their study to
preparations with high numbers of Na channels in order to improve
signal-to-noise ratios. To increase Na channel gating current
signals, single tsA201 cells (20 [mu]m in diameter) were fused into
large, multinucleated cells by treatment with polyethylene glycol
(PEG). After placement in cell culture for 48-72 hours fused tsA201
cells develop a spherical geometry with diameters up to 200?[mu]m.
Because of the large plasma membrane surface area, fused tsA201 cells
are able to express high levels of Na channels after transient
transfection with sodium channel cDNAs using Lipofectamine.
Typically, five days after transfection fused tsA201 cells that are
60 to 100?[mu]m in diameter are selected for voltage-clamp with a
large suction pipette (a pore size of 20-30?[mu]m) that allows for
both a low series resistance and internal perfusion. Approximately
two-thirds of transfected fused tsA201 cells express INa with nearly
one-third of transfected cells expressing sufficient numbers of Na
channels to allow for the ready measurement of Ig. In addition to
fused tsA201 cells being a preparation well suited for the study of
gating currents, they should also be useful for measurement of
electrical signals from other voltage-gated channels and transporters
that generate small electrical signals.
Received 2 February 1996; accepted in final form 15 April 1996.
APS Manuscript Number C65-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 28 May 96