Hi, thank you for the question.
According to this article, Mg2+ is the calcium channel blocker for TRPV6:
Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6.
Source: Journal-of-General-Physiology. [print] March 2003 2003; 121 (3): 245-260.
Publication Year: 2003
Document Type: Article-
ISSN (International Standard Serial Number): 0022-1295
"TRPV6 (CaT1/ECaC2), a highly Ca2+-selective member of the TRP
superfamily of cation channels, becomes permeable to monovalent
cations in the absence of extracellular divalent cations. The
monovalent currents display characteristic voltage-dependent gating
and almost absolute inward rectification. Here, we show that these two
features are dependent on the voltage-dependent block/unblock of the
channel by intracellular Mg2+. Mg2+ blocks the channel by binding to a
site within the transmembrane electrical field where it interacts with
permeant cations. The block is relieved at positive potentials,
indicating that under these conditions Mg2+ is able to permeate the
selectivity filter of the channel. Although sizeable outward
monovalent currents were recorded in the absence of intracellular
Mg2+, outward conductance is still apprx10 times lower than inward
conductance under symmetric, divalent-free ionic conditions. This
Mg2+-independent rectification was preserved in inside-out patches and
not altered by high intracellular concentrations of spermine,
indicating that TRPV6 displays intrinsic rectification. Neutralization
of a single aspartate residue within the putative pore loop abolished
the Mg2+ sensitivity of the channel, yielding voltage-independent,
moderately inwardly rectifying monovalent currents in the presence of
intracellular Mg2+. The effects of intracellular Mg2+ on TRPV6 are
partially reminiscent of the gating mechanism of inwardly rectifying
K+ channels and may represent a novel regulatory mechanism for TRPV6
function in vivo."
This study confirmed the above comment that MG-2+ blocked the channel
and La-3+ but 5 mu-M Ruthenium red only partially inhibited the
channel.
Biochimica-et-Biophysica-Acta. 1994; 1186 (1-2) 107-116.
Publication Year: 1994
"The present study was designed to establish the characteristics of
the Ca-2+ fluxes in isolated mitochondria of the protist Euglena
gracilis. Uptake of Ca-2+ and Sr-2+ was supported by succinate and
lactate oxidation. Ca-2+ influx was slightly inhibited by 5 mu-M
Ruthenium red and completely blocked by La-3+ with a half-maximal
inhibition attained at 50 mu-M. The addition of inorganic phosphate
induced a 3-fold stimulation of Ca-2+ uptake. Ca-2+ uptake was also
inhibited by Mg-2+."
According to this article, 5 µM Ruthenium red (cited above in a
clarification) had no effect on the channel in this test (no mention
is made of the specific channel):
A calcium conducting channel akin to a calcium pump.
Author, Editor, Inventor: Wang-Jinsong; Tang-John-M; Eisenberg-Robert-S
Author Address: Dep. Physiol., Rush Med. Coll., Chicago, Ill. 60612
Source: Journal-of-Membrane-Biology. 1992; 130 (2) 163-181.
"The calcium channels had at least three conductance states (70 pS, 50
pS and 37 pS) and were weakly selective for calcium ions, with a
permeability ratio Ca-2+ to K+ of about 3.4. The open probability of
the channel was strongly voltage dependent, decreasing at positive
membrane voltages. 10 mu-M ryanodine and 5 mu-M ruthenium red had no
effect on this channel."
----------------------------------------------------------------------------------
Additional Information:
The TRPV6 channel is regulated by Vitamin D:
Patho)physiological implications of the novel epithelial Ca2+ channels
TRPV5 and TRPV6.
Author, Editor, Inventor: Nijenhuis-Tom; Hoenderop-Joost-G-J;
Nilius-Bernd; Bindels-Rene-J-M {a}
Author Address: {a} Centrale Ontvangst Goederen, Geert Grooteplein
Zuid 30, M850 Room 07.048, 6525 GA, Nijmegen, Netherlands; E-Mail:
r.bindels@ncmls.kun.nl, Netherlands
Source: Pfluegers-Archiv-European-Journal-of-Physiology. [print] July
2003 2003; 446 (4): 401-409.
Publication Year: 2003
"The epithelial Ca2+ channels TRPV5 and TRPV6 constitute the apical
Ca2+ entry mechanism in active Ca2+ (re)absorption. These two members
of the superfamily of transient receptor potential (TRP) channels were
cloned from the vitamin-D-responsive epithelia of kidney and small
intestine and subsequently identified in other tissues such as bone,
pancreas and prostate. These channels are regulated by vitamin D as
exemplified in animal models of vitamin-D-deficiency rickets. In
addition, the epithelial Ca2+ channels might be involved in the
multifactorial pathogenesis of disorders ranging from idiopathic
hypercalciuria, stone disease and postmenopausal osteoporosis."
TRPV6 is regulated by 17beta-E2 and 1,25(OH)2D3:
American-Journal-of-Physiology. [print] July 2003 2003; 285 (1 Part 1): G78-G85.
Publication Year: 2003
Document Type: Article-
ISSN (International Standard Serial Number): 0002-9513
Language: English
"The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the
brush border membrane of intestinal cells and constitute the
postulated rate-limiting entry step of active Ca2+ absorption. The aim
of the present study was to investigate the hormonal regulation of
these channels. To this end, the effect of 17beta-estradiol
(17beta-E2), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and dietary Ca2+
on the expression of the duodenal Ca2+ transport proteins was
investigated in vivo and analyzed using real-time quantitative PCR.
Supplementation with 17beta-E2 increased duodenal gene expression of
TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase
(PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin
D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized
by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels
of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of
the stimulatory effects of 17beta-E2. Treatment with 17beta-E2
upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase
knockout mice, which was accompanied by increased serum Ca2+
concentrations from 1.69+-0.10 to 2.03+-0.12 mM (P<0.05). In addition,
high dietary Ca2+ intake normalized serum Ca2+ in these mice and
upregulated expression of genes encoding the duodenal Ca2+ transport
proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted
in increased expression of TRPV6, calbindin-D9K, and PMCA1b and
normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA
are below detection limits in these 1alpha-OHase knockout mice, but
supplementation with 1,25(OH)2D3 upregulated the expression to
significant levels. In conclusion, TRPV5 and TRPV6 are regulated by
17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved
in the regulation of TRPV6 only."
---------------------------------------------------------------------------------
Search Strategy:
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Thanks for your question. I hope this is what you are looking for. Let
me know if you need any clarification of my response.
-Anthony (adiloren-ga) |
Clarification of Answer by
adiloren-ga
on
27 May 2004 23:47 PDT
Some additional information:
In this study, ruthenium red inhibited Ca(2+) transport:
Characterization of a murine renal distal convoluted tubule cell line
for the study of transcellular calcium transport.
Characterization of a murine renal distal convoluted tubule cell line
for the study of transcellular calcium transport.
Diepens Robin J W; den Dekker Els; Bens Marcelle; Weidema A Freek;
Vandewalle Alain; Bindels Rene J M; Hoenderop Joost G J
Am J Physiol Renal Physiol 2004 Mar; 286 (3): F483-9
This article refers to ruthenium red as a "potent blocker of ECaC
activity" but notes that the ECaC2 has a low affinity for it - it also
refers to Mg(2+) as a modulator of the channel:
Function and expression of the epithelial Ca(2+) channel family:
comparison of mammalian ECaC1 and 2.
J Physiol 2001 Dec 15; 537 (Pt 3): 747-61
"The epithelial Ca(2+) channel (ECaC) family represents a unique group
of Ca(2+)-selective channels that share limited homology to the
ligand-gated capsaicin receptors, the osmolarity-sensitive channel
OTRPC4, as well as the transient receptor potential family. Southern
blot analysis demonstrated that this family is restricted to two
members, ECaC1 and ECaC2 (also named CaT1). 2. RT-PCR analysis
demonstrated that the two channels are co-expressed in
calbindin-D-containing epithelia, including small intestine, pancreas
and placenta, whereas kidney and brain only express ECaC1 and stomach
solely ECaC2. 3. From an electrophysiological point of view, ECaC1 and
ECaC2 are highly similar channels. Differences concern divalent cation
permeability, the kinetics of Ca(2+)-dependent inactivation and
recovery from inactivation. 4. Ruthenium red is a potent blocker of
ECaC activity. Interestingly, ECaC2 has a 100-fold lower affinity for
ruthenium red (IC(50) 9 +/- 1 microM) than ECaC1 (IC(50) 121 +/- 13
nM). 5. ECaCs are modulated by intracellular Mg(2+) and ATP. ECaC1 and
ECaC2 activity rapidly decay in the absence of intracellular ATP. This
effect is further accelerated at higher intracellular Mg(2+)
concentrations. 6. In conclusion, ECaC1 and ECaC2 are homologous
channels, with an almost identical pore region. They can be
discriminated by their sensitivity for ruthenium red and show
differences in Ca(2+)-dependent regulation."
Here is another study refering to ruthenium red as a calcium blocker
on the ECAC2 channel:
The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is
involved in Ca(2+)-dependent inactivation.
Pflugers Arch 2003 Feb; 445 (5): 584-8
"The family of epithelial Ca(2+) channels (ECaC) is a unique group of
highly Ca(2+)-selective channels consisting of two members, ECaC1 and
ECaC2. We used carboxyl terminal truncations and mutants to delineate
the molecular determinants of the Ca(2+)-dependent inhibition of ECaC.
To this end, rabbit ECaC1 was expressed heterologously with green
fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells
using a bicistronic vector. Deletion of the last 30 amino acids of the
carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity
significantly. Another critical sequence for Ca(2+)-dependent
inactivation of ECaC1 was found upstream in the carboxyl terminus.
Analysis of truncations at amino acid 635, 639, 646, 649 and 653
disclosed a critical sequence involved in Ca(2+)-dependent
inactivation at positions 650-653. C653X showed decreased Ca(2+)
sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent
inactivation. Interestingly, the number of green fluorescent cells,
which is an index of the number of transfected cells, was
significantly smaller for cells transfected with truncations shorter
than E649 than for cells transfected with wild-type ECaC. However, the
expression level of GFP was restored in the presence of the ECaC
blocker ruthenium red, suggesting that these truncations resulted in
deleterious Ca(2+) influx. In conclusion, we have identified two
domains in the carboxyl terminus of ECaC1 that control
Ca(2+)-dependent inactivation."
The full text of this article may be useful:
ECaC: the gatekeeper of transepithelial Ca2+ transport. (48 REFS)
Biochim Biophys Acta 2002 Nov 4; 1600 (1-2): 6-11
"The epithelial Ca(2+) channels (ECaCs) are primarily expressed in
Ca(2+) transporting epithelia and represent a new family of Ca(2+)
channels that belong to the superfamily of transient receptor
potential (TRP) channels. Two members, namely ECaC1 and ECaC2, have
been identified from kidney and intestine, respectively. These
channels are the prime target for hormonal control of active Ca(2+)
flux from the urine space or intestinal lumen to the blood
compartment. This review covers the distinctive properties of these
highly Ca(2+)-selective channels and highlights the implications for
our understanding of the process of transepithelial Ca(2+) transport."
"Transcellular Ca(2+) transport across mpkDCT cells was completely
inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not
by the voltage-operated Ca(2+) channel inhibitors felodipine and
verapamil. With the use of patch-clamp analysis, the IC(50) of
ruthenium red on Na(+) currents was between the values measured for
TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5
and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in
combination with IBMX, 1,25-dihydroxyvitamin D(3), and
1-deamino-8-d-arginine vasopressin increased transcellular Ca(2+)
transport, whereas PMA and parathyroid hormone had no significant
effect. In conclusion, the murine mpkDCT cell line provides a unique
cell model in which to study the molecular regulation of transcellular
Ca(2+) transport in the kidney in vitro."
In this study thapsigargin was used as a calcium inhibitor:
Store-operated Ca2+ current and TRPV6 channels in lymph node prostate cancer cells.
J Biol Chem 2003 Dec 19; 278 (51): 50872-9
"The contribution of endogenous and recombinant transient receptor
potential vanilloid type 6 (TRPV6) channels to Ca2+ entry across the
plasma membrane was studied in the human lymph node prostate cancer
cell line (LNCaP). LNCaP cells do express the TRPV6 gene, and Ca2+
entry currents in these cells were detected after active and passive
Ca2+ store depletion by intracellular application of inositol
1,4,5-trisphosphate, Ca2+ chelators, and the sarcoplasmic/endoplasmic
reticulum Ca2+-ATPase inhibitor thapsigargin."
CaT1 knock-down strategies fail to affect CRAC channels in
mucosal-type mast cells.
Kahr Heike; Schindl Rainer; Fritsch Reinhard; Heinze Barbara; Hofbauer
Michael; Hack Marlene E; Mortelmaier Manuel A; Groschner Klaus; Peng
Ji-Bin; Takanaga Hitomi; Hediger Matthias A; Romanin Christoph
ORGA: Institute for Biophysics, University of Linz, Altenbergerstr.
69, A-4040 Linz, Austria. Email: christoph.romanin@jku.at
CITE: J Physiol 2004 May 15; 557 (Pt 1): 121-32
"CaT1, the calcium transport protein 1 encoded by TRPV6, is able to
generate a Ca(2+) conductance similar but not identical to the
classical CRAC current in mucosal-type mast cells. Here we show that
CaT1-derived Ca(2+) entry into HEK293 cells is effectively inhibited
either by expression of various dominant negative N-terminal fragments
of CaT1 (N(334)-CaT1, N(198)-CaT1 and N(154)-CaT1) or by antisense
suppression. By contrast, the endogenous CRAC current of the mast
cells was unaffected by CaT1 antisense and siRNA knockdown but
markedly suppressed by two (N(334)-CaT1, N(198)-CaT1) of the dominant
negative N-CaT1 fragments. Inhibition of CRAC current was not an
unspecific, toxic effect, as inward rectifier K(+) and MagNuM currents
of the mast cells were not significantly affected by these N-CaT1
fragments. The shortest N(154)-CaT1 fragment inhibited CaT1-derived
currents in mast cells, but failed to inhibit CRAC currents. Thus, the
structural requirements of rCaT N-terminal fragments for inhibition of
rCaT1 and CRAC channels are different. These results together with the
lack of CaT1 antisense and siRNA effects on currents render it
unlikely that CaT1 is a component of native CRAC channels in mast
cells. The data further demonstrate a novel strategy for CRAC current
inhibition by an N-terminal structure of CaT1."
This article confirms that TRPV6 and TRPV5 are important channels for
calcium transport and absorption:
Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in
mice lacking TRPV5.
Hoenderop Joost G J; van Leeuwen Johannes P T M; van der Eerden Bram C
J; Kersten Ferry F J; van der Kemp Annemiete W C M; Merillat
Anne-Marie; Waarsing Jan H; Rossier Bernard C; Vallon Volker; Hummler
Edith; Bindels Rene J M
J Clin Invest 2003 Dec; 112 (12): 1906-14
"Ca2+ ions play a fundamental role in many cellular processes, and the
extracellular concentration of Ca2+ is kept under strict control to
allow the proper physiological functions to take place. The kidney,
small intestine, and bone determine the Ca2+ flux to the extracellular
Ca2+ pool in a concerted fashion. Transient receptor potential (TRP)
cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have
recently been postulated to be the molecular gatekeepers facilitating
Ca2+ influx in these tissues and are members of the TRP family, which
mediates diverse biological effects ranging from pain perception to
male aggression. Genetic ablation of TRPV5 in the mouse allowed us to
investigate the function of this novel Ca2+ channel in maintaining the
Ca2+ balance. Here, we demonstrate that mice lacking TRPV5 display
diminished active Ca2+ reabsorption despite enhanced vitamin D levels,
causing severe hypercalciuria. In vivo micropuncture experiments
demonstrated that Ca2+ reabsorption was malfunctioning within the
early part of the distal convolution, exactly where TRPV5 is
localized. In addition, compensatory hyperabsorption of dietary Ca2+
was measured in TRPV5 knockout mice. Furthermore, the knockout mice
exhibited significant disturbances in bone structure, including
reduced trabecular and cortical bone thickness. These data demonstrate
the key function of TRPV5 in active Ca2+ reabsorption and its
essential role in the Ca2+ homeostasis."
Please let me know if this is an appropriate response to your
question. I am not overly famiar with this concentration within the
field and may require some further information. I hope this
information and the information provided in the above answer is
helpful. Thanks again for your question.
-Anthony
|
Clarification of Answer by
adiloren-ga
on
27 May 2004 23:56 PDT
Sorry- the excerpt from the first source --
Characterization of a murine renal distal convoluted tubule cell line
for the study of transcellular calcium transport.
Diepens Robin J W; den Dekker Els; Bens Marcelle; Weidema A Freek;
Vandewalle Alain; Bindels Rene J M; Hoenderop Joost G J
Am J Physiol Renal Physiol 2004 Mar; 286 (3): F483-9
was mistakenly pasted below the excerpt --
ECaC: the gatekeeper of transepithelial Ca2+ transport. (48 REFS)
Biochim Biophys Acta 2002 Nov 4; 1600 (1-2): 6-11
here is the full excerpt:
Am J Physiol Renal Physiol 2004 Mar; 286 (3): F483-9
"To unravel the molecular regulation of renal transcellular Ca(2+)
transport, a murine distal convoluted tubule (mpkDCT) cell line
derived from distal convoluted tubules (DCT) microdissected from a
SV-PK/Tag transgenic mouse was characterized. This cell line
originated from DCT only, as mRNA encoding for the DCT marker
thiazide-sensitive Na(+)/Cl(-) cotransporter was expressed, whereas
mRNA encoding for the connecting tubule and collecting duct marker
aquaporin-2 was not detected, as determined by reverse-transcriptase
PCR. mpkDCT cells expressed mRNA encoding the Ca(2+) channels TRPV5
and TRPV6 and other key players necessary for transcellular Ca(2+)
transport, i.e., calbindin-D(9k), calbindin-D(28k), plasma membrane
Ca(2+)-ATPase isoform 1b, and Na(+)/Ca(2+) exchanger 1. Primary
cultures of DCT cells exhibited net transcellular Ca(2+) transport of
0.4 +/- 0.1 nmol.h(-1).cm(-2), whereas net transcellular Ca(2+)
transport across mpkDCT cells was significantly higher at 2.4 +/- 0.4
nmol.h(-1).cm(-2). Transcellular Ca(2+) transport across mpkDCT cells
was completely inhibited by ruthenium red, an inhibitor of TRPV5 and
TRPV6, but not by the voltage-operated Ca(2+) channel inhibitors
felodipine and verapamil. With the use of patch-clamp analysis, the
IC(50) of ruthenium red on Na(+) currents was between the values
measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting
that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin
in combination with IBMX, 1,25-dihydroxyvitamin D(3), and
1-deamino-8-d-arginine vasopressin increased transcellular Ca(2+)
transport, whereas PMA and parathyroid hormone had no significant
effect. In conclusion, the murine mpkDCT cell line provides a unique
cell model in which to study the molecular regulation of transcellular
Ca(2+) transport in the kidney in vitro."
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