Cardiovascular Journal of Africa: Vol 27 No 1 (January/February 2016)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 1, January/February 2016 AFRICA 5
macromolecular complexes and pathways involved. Taking
into consideration the relevance of KCNE2 in the context of
ion channel regulation and LQTS, this study aimed to identify
interactors with this β-subunit; specifically focusing on its
cytoplasmic C-terminal domain, for which functional roles
remain inadequately described.
Using yeast two-hybrid analysis, we identified filamin C
(FLNC) as aKCNE2-interacting protein. FLNC and its paralogs,
filamin A (FLNA) and filamin B (FLNB), act as scaffolding
proteins and have been implicated in a number of cellular stress
responses,32-38 including several hypoxia-related effects.35–38 For
this reason, co-localisation and co-immunoprecipitation (Co-IP)
analyses for verification of this interaction were conducted both
under normoxic and hypoxic conditions.
Here, we show that, under normoxic and hypoxic conditions,
FLNC and KCNE2c co-localised within the cell. However,
FLNC and KCNE2 only co-immunoprecipitated under hypoxic
conditions, suggesting that while these two proteins are located
in close proximity to one another within the cell, it is only
under conditions of cellular stress that a physical interaction
between the two exists. The data presented here provide evidence
to suggest that KCNE2 may play a role in hypoxia-induced
arrhythmias.
Methods
KCNE2 construct
A fragment encoding the C-terminal of KCNE2 gene (amino acid
72-123) was amplified from human genomic DNA by means of
polymerase chain reaction (PCR). The PCR reaction employed
KCNE2 C-terminal-specific primers with two restriction enzyme
sites (NdeI and Eco R1) (Table 1) for subsequent cloning into
the CLONTECH yeast two-hybrid (Y2H) bait vector, pGBKT7
(pGBKT7-KCNE2), in-frame with the GAL4-DNA binding
domain (GAL4BD). The integrity of the sequence and the
conservation of the GAL4 domain reading frame of the resulting
construct were verified via sequencing.
Yeast two-hybrid (Y2H) library screen
The Saccharomyces cerevisiae strain, AH109 (BD Biosciences,
Clontech, USA), was transformed with the pGBKT7-KCNE2
construct and mated with the S cerevisiae strain, Y187, which
was pre-transformed with a MATCHMAKER human cardiac
cDNA library (BD Biosciences, Clontech, USA). Subsequently,
the library screen was conducted according to manufacturer’s
recommendations.
The prey plasmids, from colonies expressing the three essential
reporter genes (HIS3, ADE2 and MEL1), were isolated from
the diploid yeast cells and were retransformed into S cerevisiae
strain Y187 to analyse their ability to activate the reporter genes
when mated with heterologous baits (Table 2). Prey peptides
showing specific interaction with the KCNE2 C-terminal domain
were sequenced and the in-frame open reading frame (ORF)
sequences were analysed using BLASTN and BLASTP against
public databases .
Cell culture
The H9C2 rat-derived cardiac myoblasts (American Typer
Culture Collection, USA) were grown in Dulbecco’s modified
Eagle medium (DMEM, Lonza, CHE) containing 10% foetal
bovine serum (FBS, Biochrom, GER) and 1% penicillin/
streptomycin (Pen/Strep, Biochrom, GER) until they reached
80% confluency. For co-localisation, 10 000 cells were seeded
onto glass cover slips in each well of a six-well plate (8-cm2
culture dishes) and incubated until 80% confluency was reached,
while for Co-IP, cells were grown in 175-cm2 flasks until
they reached 80% confluency. Differentiation medium (DMEM
containing 1% horse serum and 1% Pen/Strep) was subsequently
added to each well of the six-well plate and the 175-cm 2 flasks.
Cells were differentiated for 10–14 days.
For hypoxia induction, the differentiation medium was
removed and replaced with Esumi buffer (138.6 mM NaCl, 12
mM KCl, 1 mM MgCl2, 1 mM CaCl2.H2O, and 4 mM Hepes,
pH 6.2).39 Culture dishes and flasks were then placed in a
chamber where a hypoxic environment was created by flushing
the system with a 1% O2 gas mixture at a flow rate of 20 l/min,
for approximately four minutes. The cells were then incubated in
the hypoxic chamber at 37°C for two hours.
For Co-IP experiments, 5 ml of pre-warmed trypsin was
used to detach the cells from the growth surface of the flasks.
The cells were then centrifuged at 4ºC for three minutes at 2 500
rpm. The supernatant was discarded and the pellet resuspended
in 1 ml of phosphate-buffered saline (PBS) and re-pelleted at 9
000 rpm for two minutes. The PBS was removed and the cells
were then lysed with ice-cold lysis buffer (50 mM Hepes, 5 M
NaCl, 0.5 M EDTA, 1% Triton X-100, 1 M Na3VO4) containing
protease inhibitor cocktail tablets [one tablet EDTA-free protease
inhibitor cocktail tablet per 20 ml lysis buffer and 1 mM
phenylmethylsulfonylfluoride (PMSF) (Sigma-Aldrich, USA)].
Approximately 0.5 ml of ZROB05 Ceria zirconium oxide
beads (0.5 mm diameter) (Next Advance Inc, USA) was added
to the suspension and it was placed in a Bullet blender® (Gentaur,
GBR) for one minute. The blending step was repeated three
times at five-minute intervals. The cells were then pelleted by
centrifugation at 9 000 rpm for two minutes, after which the
supernatant was collected. A Bradford assay was used for protein
Table 1. Nucleotide sequences of primers used
to amplify the C-terminal of KCNE2
Primer Sequence (5’-3’) Ta (°C)
KCNE2-
forwardNde1
5’ - ACTGCAGAACATATGCTCAAATCCAAGAGA-
CGG - 3’ 50
KCNE2-
reverseEcoR1
5’ - ACTGCAGAAGAATTCCTA TCAGGGGAA-
CATTTTGAAC - 3’ 51
°C: degrees Celsius; Ta: annealing temperature; KCNE2: potassium voltage-
gated ion-channel subfamily E member 2.
The bold text represents a tag, which facilitates restriction enzyme digestion,
while the underlined sequences correspond to the Nde1 and EcoR1 restriction
enzyme sites, respectively. The short italic sequence (CTA) symbolises the stop
codon, and the remaining text represents the sequence of the primer, which will
anneal to the DNA in the PCR amplification reaction.
Table 2. S cerevisiae bait strains
S cerevisiae bait strains Plasmid type
AH109 pGBKT7-KCNE2 Positive control plasmid
AH109 pGBKT7 Non-recombinant plasmid
AH109 pGBKT-53* Control bait plasmid
AH109 pGBKT7-WFS1 Negative control plasmid
*The pGBKT7 vector containing the humanp53 gene. KCNE2: potassium
voltage-gated ion-channel subfamily E member 2; WFS1: Wolfram syndrome 1.