Hamilton, Kirk L.

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Kirk L. Hamilton

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BLAP-Tagged Channel Technology: A New Direction Trafficking toward Epithelial Physiology

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Frontiers in Physiology

www.frontiersin.org December 2010 | Volume 1 | Article 162 | 1
General Commentary
published: 13 December 2010
doi: 10.3389/fphys.2010.00162
BLAP-tagged channel technology: a new direction trafficking 
toward epithelial physiology
Kirk L. Hamilton*
Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
*Correspondence: kirk.hamilton@otago.ac.nz
A commentary on
Immunofluorescence-based assay to iden-
tify modulators of the number of plasma 
membrane KCa3.1 channels.
by Balut, C. M., Gao, Y., Luke, C., and 
Devor, D. C. (2010). Future Med. Chem. 2, 
707–713.
Recent developments in fluorescent-probe 
technology have allowed real-time imaging 
of cellular events by engineering small pep-
tide sequences [e.g., 15 amino acid sequence 
for a biotin-ligase acceptor peptide (BLAP), 
see Chen et al., 2005; Howarth and Ting, 
2008] in an external domain of a pro-
tein (e.g., ion channel). The BLAP-tagged 
channels are biotinylated at the membrane 
with recombinant BirA (a biotin ligase 
from Escherichia coli) and labeled with a 
 streptavidin–fluorophore, after which, the 
membrane and cellular fates of the BLAP-
tagged channels can be determined. The 
labeling of the BLAP-tagged channels is 
relatively rapid (less than 50 min), very 
efficient (covalent binding), sensitive, and 
effective for protein detection by immun-
ofluorescence and Western blot (Chen et al., 
2005; Howarth and Ting, 2008).
Recently, Devor and colleagues (Balut 
et al., 2010b; Gao et al., 2010), using the 
BLAP-technology based on work of Ting, 
have defined the membrane residence and 
retrograde trafficking of the Ca2+-activated 
K+ channels, KCa3.1 and KCa2.3, in human 
embryonic kidney (HEK293) cells and 
endothelial cells (HMEC-1). The engineer-
ing of the BLAP-tag within both KCa3.1 
and KCa2.3 did not alter gating or Ca2+-
dependence of the channels (Gao et al., 
2010). Further, Gao et al. (2010) demon-
strated that KCa2.3 had a long membrane 
residence time (∼13 h) and that the channel 
was recycled back to the membrane, while 
KCa3.1 had a short membrane residence 
time (∼ 90 min), and was destined for the 
lysosomes (Balut et al., 2010b).
Both KCa3.1 and KCa2.3 have been 
suggested as therapeutic targets in many 
diseases including endothelial dysfunction 
and hypertension (Kohler et al., 2010). 
Pharmacological modulation and/or alter-
ing membrane channel residence time of 
these channels have promise as potential 
therapeutic targets for ameliorating diseases 
(Kohler et al., 2010). The question is how 
does one quickly screen potential modula-
tors of residence time and endocytosis of a 
channel in a membrane?
The focus of this Commentary is to 
highlight recent work of Devor and col-
leagues (Balut et al., 2010a) in which they 
developed a novel immunofluorescent-
based assay to assess modulators of endo-
cytosis of proteins (e.g., KCa3.1). With this 
technique, they were able to quickly deter-
mine the channel membrane residence 
time and rate of endocytosis of KCa.1. 
This establishes a crucial paradigm for the 
further investigation of this channel, and 
other membrane proteins, as a therapeutic 
target(s) for clinical use.
Initially, Devor and colleagues realized 
the time of labeling of KCa3.1 must be 
reduced in order to rapidly assess modu-
lators of channel endocytosis, and for the 
effectiveness of their procedure to be func-
tionally viable in a 96-well plate format assay 
approach with future application in a high-
throughput scenario. Indeed, they reduced 
the time of labeling KCa3.1 from 50 min to 
less than 15 min. This was accomplished by 
subcloning BLAP-tagged KCa3.1 (KCa3.1-
BLAP) and BirA [with a KDEL endoplas-
mic reticulum (ER) retention motif] into a 
bicistronic plasmid. Following this, a stable 
HEK293 cell line was established expressing 
both the BLAP-tagged KCa3.1 and BirA-
KDEL. So, as each channel subunit is synthe-
sized it is also biotinylated, providing a high 
efficiency of biotinylation. This molecular 
engineering innovation greatly reduced the 
time of labeling and the increased efficiency 
of biotinylation the channel.
Devor and colleagues (Balut et al., 2010a) 
have provided exciting experimental data, 
and “proof of concept” of an immun-
ofluorescence-based assay in which they 
screened a modulator of the endocytosis 
of KCa3.1-BLAP. The overall experimental 
paradigm was to label membrane surface 
KCa3.1-BLAP and total plasma membrane 
with different fluorophores to determine 
the intracellular/membrane location of 
the channel and determine the effects of 
inhibition of the endocytotic pathway on 
the membrane residence time of KCa3.1-
BLAP. The evidence for endocytosis of 
KCa3.1-BLAP was assessed by the absence 
of membrane colocalization of the fluores-
cent signals (i.e., endocytosis).
Briefly, experiments were conducted in a 
96-well plate format in which, surface chan-
nels and membranes of control cells were 
labeled at 4°C to prevent endocytosis of 
the channel during the labeling procedure. 
Additionally, for experimental cells, only 
surface channels were labeled (4°C) and 
were placed at 37°C for assessing channel 
membrane residence time and endocyto-
sis. After 90 min, experimental cells, pre-
labeled KCa3.1-BLAP, were then labeled at 
4°C with a different fluorophore specific 
for the membrane and colocalization was 
assessed. This was an adaptation required 
for the use of an automated analysis system 
and to assure that only plasma membrane 
was labeled prior to fixation and permea-
bilization of the cells (Balut et al., 2010a). 
Balut et al. (2010a) clearly demonstrated 
that at time 0, KCa3-BLAP was only present 
at the membrane with robust colocaliza-
tion of fluorescent labeling and that chan-
nels were internalized (i.e., within vesicles) 
within 90 min.
In order to assess endocytosis of KCa3.1-
BLAP, Devor and colleagues incubated cells 
expressing KCa3.1-BLAP with UBEI-41, an 
inhibitor of ubiquitin-activating enzyme E; 
which blocks the first step of the ubiquitina-
tion pathway. Therefore, at time 0, control 
Frontiers in Physiology | Renal and Epithelial Physiology  December 2010 | Volume 1 | Article 162 | 2
Hamilton BLAP-tagged channels
I thank Drs Daniel C. Devor and Corina M. 
Balut for further clarification of experimen-
tal details that were not included in their 
paper (Balut et al., 2010a). This work was 
supported by a NZ Lottery Board grant and 
the Department of Physiology, University 
of Otago.
RefeRences
Balut, C. M., Gao, Y., Luke, C., and Devor, D. C. (2010a). 
Immunofluorescence-based assay to identify modu-
lators of the number of plasma membrane KCa3.1 
channels. Future Med. Chem. 2, 707–713.
Balut, C. M, Gao, Y., Murray, S. A., Thibodeau, P. H., 
and Devor, D. C. (2010b). ESCRT-dependent tar-
geting of plasma membrane localized KCa3.1 to 
the lysosomes. Am. J. Physiol., Cell Physiol. 299, 
C1015–C1027.
Chen, I., Howarth, M., Lin, Y., and Ting, A. Y. (2005). 
Site-specific labeling of cell surface proteins with 
biophysical probes using biotin ligase. Nat. Methods 
2, 99–104.
Gao, Y., Balut, C. M., Bailey, M. A., Patino-Lopez, G., 
Shaw, S., and Devor, D. C. (2010). Recycling of the 
Ca2+-activated K+ channel, KCa2.3, is dependent upon 
RME1, Rab35/EPI64C, and an N-terminal domain. J. 
Biol. Chem. 285, 17938–17953.
Howarth, M., and Ting, A. Y. (2008). Imaging proteins in 
live mammalian cells with biotin ligase and monova-
lent streptavidin. Nat. Protoc. 3, 534–545.
Kohler, R., Kaistha, B. P., and Wulff, H. (2010). Vascular 
K
Ca
-channels as therapeutic targets in hypertension 
and restenosis disease. Expert Opin. Ther. Targets 14, 
143–155.
Received: 20 October 2010; accepted: 29 November 2010; 
published online: 13 December 2010.
Citation: Hamilton KL (2010) BLAP-tagged channel tech-
nology: a new direction trafficking toward epithelial physiol-
ogy. Front. Physio. 1:162. doi: 10.3389/fphys.2010.00162
This article was submitted to Frontiers in Renal and Epithelial 
Physiology, a specialty of Frontiers in Physiology.
Copyright © 2010 Hamilton. This is an open-access arti-
cle subject to an exclusive license agreement between the 
authors and the Frontiers Research Foundation, which 
permits unrestricted use, distribution, and reproduction 
in any medium, provided the original authors and source 
are credited.
immunofluorescence assay to examine the 
potential modulators of KCa2.3. Additionally, 
Devor is developing an assay, using KCa3.1-
BLAP technology, to examine the trafficking 
of the channel from the ER to the plasma 
membrane. This assay will certainly occupy a 
unique niche for advancing our understand-
ing of anterograde trafficking of ion chan-
nels (and other proteins), and prove useful in 
determining the fate of membrane proteins 
targeted specifically to the apical or basola-
teral membrane of polarized epithelia.
Why is this papeR impoRtant foR 
epithelial physiologists?
We need to understand the basic trafficking 
of epithelial ion channels to determine the 
possible role(s) of ion channels in diseases. 
The novel immunofluorescent-tagging 
approach that Devor and colleagues have 
developed will undoubtedly foster an excit-
ing research avenue for epithelial physiology. 
Devor and colleagues have clearly provided 
an assay that can identify “small-molecule 
modulators” of endocytosis that lead to 
increased numbers of channels at the mem-
brane, and thus, can enhance a physiological 
response (Balut et al., 2010a). Certainly, we 
must keep our “research eyes” open for novel 
technologies that can be widely applied not 
just by epithelial physiologists, but also by 
many molecular cell physiologists to further 
our understanding of ion channel physiol-
ogy. Exciting possibilities exist for those 
willing to take the “BLAP-tag leap” into a 
new technology.
acknoWledgments
I thank Drs Chris P. Bolter and Marcelo 
Carattino for critically reading this manu-
script and for many helpful comments. 
cells were either labeled as described above, 
or only surface channels of experimental 
cells were labeled and placed at 37°C for 
90 min and incubated in the presence or 
absence of the UBEI-41. Again, after 90 min, 
membrane proteins were labeled at 4°C. In 
the presence UBEI-41, KCa3.1-BLAP was 
retained at the membrane with similar colo-
calization of fluorescent signal as compared 
to control cells (i.e., only surface channels). 
Here, Devor and colleagues demonstrated 
they could capture a “snapshot” of traf-
ficking of KCa3.1-BLAP and identify an 
inhibitor of endocytosis of KCa3.1-BLAP 
in a 96-well plate format. For experimental 
and analysis details, the reader is directed 
to Balut et al. (2010a); which will be freely 
available in April 2011.
The immunofluorescence assay described 
by Devor and colleagues has many desirable 
aspects that will contribute to advances in 
the trafficking physiology of channels. First, 
with this assay, the time for labeling of the 
channel is greatly reduced from 50 min 
down to less than 15 min. This has fur-
ther implications of saving time and cost 
of materials when screening hundreds of 
wells for a single experiment. Secondly, their 
experimental approach achieved high effi-
ciency of biotinylation as the ER-retained 
biotin ligase biotinylates each channel 
subunit as it is synthesized. Finally, the 
immunofluorescence assay provides “proof 
of concept” that this paradigm can be used 
in high-throughput screening strategy for 
identifying potential small-molecule mod-
ulators of endocytosis of specific proteins 
(e.g., channels) of the plasma membrane.
Currently, Devor and colleagues (personal 
communication) are modifying the experi-
mental design of the 96-well plate format 


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BLAP-Tagged Channel Technology: A New Direction Trafficking toward Epithelial Physiology

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