Please cite this article in press as: M. Irnaten, et al., Rapid effects of 17-estradiol on epithelial TRPV6 Ca
2+
channel in
human T84 colonic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2008.02.007
ARTICLE IN PRESS
+Model
YCECA-987; No. of Pages 12
Cell Calcium (2008) xxx, xxx—xxx
journal homepage: www.elsevier.com/locate/ceca
Rapid effects of 17-estradiol on epithelial TRPV6
Ca
2+
channel in human T84 colonic cells
Mustapha Irnaten
*,1
, Nicolas Blanchard-Gutton
1
, Brian J. Harvey
Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland
Received 22 May 2007; received in revised form 18 January 2008; accepted 12 February 2008
KEYWORDS
Non-genomic;
17-Estradiol;
TRPV6;
Calcium;
T84 colonic cells
Summary The control of calcium homeostasis is essential for cell survival and is of crucial
importance for several physiological functions. The discovery of the epithelial calcium channel
Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca
2+
absorp-
tive pathways involved in the regulation of whole body Ca
2+
homeostasis. The role of steroid
hormone 17-estradiol (E
2
), in [Ca
2+
]
i
regulation involving TRPV6 has been only limited at the
protein expression levels in over-expressing heterologus systems. In the present study, using
a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology
to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is
natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic
cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the
rapid effects of E
2
in [Ca
2+
]
i
regulation involving directly TRPV6 channels in T84 cells. Treat-
ment with E
2
rapidly (<5 min) enhanced [Ca
2+
]
i
and this increase was partially but significantly
prevented when cells were pre-treated with ruthenium red and completely abolished in cells
treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that
when cells are stimulated by E
2
, Ca
2+
enters the cell through TRPV6 channels. TRPV6 channels
in T84 cells contribute to the Ca
2+
entry/signalling pathway that is sensitive to 17-estradiol.
© 2008 Elsevier Ltd. All rights reserved.
Introduction
Calcium is one of the most abundant cation in the human
body and it is required for many important physiological
processes. The Ca
2+
concentration in blood and extracel-
∗
Corresponding author. Department of Molecular Medicine, Royal
College of Surgeons in Ireland, Beaumont Hospital, PO Box 9063,
Dublin 9, Ireland. Tel.: +353 1 809 3825; fax: +353 1 809 3778.
E-mail address: mirnaten@rcsi.ie (M. Irnaten).
1
Both authors contributed equally to this work.
lular fluids is dependent upon the balance of movement of
Ca
2+
via intestinal absorption, bone formation or breakdown,
and kidney reabsorption. As all of the Ca
2+
in the body is
obtained from the diet through gastrointestinal absorption,
the regulation of intestinal Ca
2+
absorption is crucial for
maintaining whole body calcium homeostasis [1]. The human
T84 colonic epithelial cell line is a well-characterized tissue
culture model of intestinal ion transport and is responsive
to hormones involved in the regulation of Ca
2+
-dependent
absorptive processes.
Many hormones, neurotransmitters and physical stimuli
elicit cellular responses through changes in the levels of the
0143-4160/$ — see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2008.02.007