1924. |
Direct Observation of Individual KCNQ1 Potassium Channels Reveals Their Distinctive Diffusive Behavior
Gregory I. Mashanov, Muriel Nobles, Stephen C. Harmer, Justin E. Molloy, and Andrew Tinker,
Medical Research Council National Institute for Medical Research,
Journal of Biological Chemistry,
285(1924),
(2010)
Link To Paper
Materials & Methods:
HEK293 cells were cultured and transfected with Lipofectamine
or Lipofectamine 2000 as previously described (22).
The HL-1 cells were a generous gift from Prof. William C. Claycomb
and were grown in Claycomb medium supplemented
with 10% fetal bovine serum (Sigma), 2 mM L-glutamine, 100
units/ml of penicillin, 100 g/ml of streptomycin, and 0.1 mM
norepinephrine (Sigma). The medium was replaced every day,
and cells were grown on T75 flasks pre-coated with gelatin/
fibronectin. For TIRF microscopy and confocal imaging, HL-1
cells were seeded at a lower density (1:20) on 25-mm glass coverslips
(BDH) or glass-bottom Petri dishes (Mattek). HL-1 cells
were transfected using FuGENE HD transfection reagent
(Roche Applied Science). Cells were plated 48–72 h before
transfection. The transfection complex contained 97 l of Claycomb
medium (no supplement), 5 l of FuGENE HD, and the
DNA. The transfection complex was added to the cell culture
and left for 24 h. Experiments were performed 48 h after transfection.
Under our standard transfection conditions with cells
in 35-mm dishes we used 800–1600 ng of DNA for both cell types. However, in single molecule experiments with TIRFM
we titrated this downwards to 25–160 ng in total (25–80 ng of
each individual construct for cotransfections but generally 40
ng for each construct) for both cell types. This led to high efficiency
transfection but relatively low-level protein expression
in individual cells and so made single-particle tracking easier.
The plasmid expressing KCNE1 is smaller ( 5 kb) than that
expressing KCNQ1-GFP ( 8 kb) thus the molar ratio
approached 1:2. Microscopic Technique
Laser Scanning Confocal Microscopy Cell Type(s)
HL-1 cells |
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glass-bottom Petri dishes (Mattek) 