13 research outputs found
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore
Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore
Expression of TRPC6 channels in human epithelial breast cancer cells
<p>Abstract</p> <p>Background</p> <p>TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.</p> <p>Methods</p> <p>Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).</p> <p>Results</p> <p>A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La<sup>3+</sup>. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.</p> <p>Conclusion</p> <p>Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.</p
Exploring the Therapeutic Potential of Membrane Transport Proteins: Focus on Cancer and Chemoresistance
Improving the therapeutic efficacy of conventional anticancer drugs represents the best hope for cancer treatment. However, the shortage of druggable targets and the increasing development of anticancer drug resistance remain significant problems. Recently, membrane transport proteins have emerged as novel therapeutic targets for cancer treatment. These proteins are essential for a plethora of cell functions ranging from cell homeostasis to clinical drug toxicity. Furthermore, their association with carcinogenesis and chemoresistance has opened new vistas for pharmacology-based cancer research. This review provides a comprehensive update of our current knowledge on the functional expression profile of membrane transport proteins in cancer and chemoresistant tumours that may form the basis for new cancer treatment strategies
Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel
Opening and closing of the cystic fibrosis transmembrane
conductance
regulator chloride channel are controlled by interactions of ATP with
its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected
to the transmembrane pore via four cytoplasmic loops. These loops
have been suggested to play roles both in channel gating and in forming
a cytoplasmic extension of the channel pore. To investigate the structure
and function of one of these cytoplasmic loops, we have used patch
clamp recording to investigate the accessibility of cytoplasmically
applied cysteine-reactive reagents to cysteines introduced into loop
3. We find that methanethiosulfonate (MTS) reagents modify cysteines
introduced at 14 of 16 sites studied in the juxtamembrane region of
loop 3, in all cases leading to inhibition of channel function. In
most cases, both the functional effects of modification and the rate
of modification were similar for negatively and positively charged
MTS reagents. Single-channel recordings indicated that, at all sites,
inhibition was the result of an MTS reagent-induced decrease in channel
open probability; in no case was the Cl<sup>–</sup> conductance
of open channels altered by modification. These results indicate that
loop 3 is readily accessible to the cytoplasm and support the involvement
of this region in the control of channel gating. However, our results
do not support the hypothesis that this region is close enough to
the Cl<sup>–</sup> permeation pathway to exert any influence
on permeating Cl<sup>–</sup> ions. We propose that either the
cytoplasmic pore is very wide or cytoplasmic Cl<sup>–</sup> ions use other routes to access the transmembrane pore
Activation of the calcium-sensing receptor by high calcium induced breast cancer cell proliferation and TRPC1 cation channel over-expression potentially through EGFR pathways
International audienceThe calcium sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium ([Ca(2+)](o)). In MCF-7 human breast cancer cells, we previously reported that treatment with [Ca(2+)](o) for 24h leads to an over-expression of the Transient Receptor Potential Canonical 1 (TRPC1) cation channel and cell proliferation. Both involve the extracellular signal-regulated Kinases 1 & 2 (ERK1/2). MCF-7 also expressed epidermal growth factor receptor (EGFR) which is involved in cell proliferation through ERK1/2. Therefore, we investigated the cross-talk between CaR and EGFR in mediating ERK1/2 phosphorylation, TRPC1 over-expression and cell proliferation. Our data show that both high [Ca(2+)](o) and EGF phosphorylate ERK1/2. Furthermore, inhibition of EGFR kinase and matrix metalloproteinases (MMPs) reduced the overall effects mediated by [Ca(2+)](o) such as activation of ERK1/2, expression of TRPC1 and cell proliferation. They indicate the important role of the CaR-EGFR-ERK axis in transmitting mitogenic signals generated by high [Ca(2+)](o) in MCF-7 cells
Expression of K+ channels in normal and cancerous human breast
Potassium (K+) channels contribute to the
regulation of cell proliferation and apoptosis and are also
involved in tumor generation and malignant growth.
Using immunohistochemical analysis, we
investigated the expression of four K+ channels GIRK1
(G-Protein Inwardly Rectifying Potassium Channel 1),
Ca2+-activated K channel (KCa1.1), voltage activated K+
channels (KV 1.1 and KV 1.3) and of the anti-apoptotic
protein Bcl2 in normal and cancerous breast tissues and
compared their expression with clinicopathological data.
GIRK1 was overexpressed in carcinomatous tissues. In
contrast, KV 1.1 and KV 1.3 were less expressed in
cancerous tissue. The expression of Bcl-2 was similar in
both tissues. As to the clinicopathological data, a
correlation between KCa1.1 channel and estrogen
receptor (ER) expression was observed.
GIRK1 was overexpressed in breast carcinoma
suggesting its involvement in proliferation and
oncogenesis and its possible use as a putative
pharmaceutigal target. The correlation between KCa1.1
channel and ER suggests the involvement of this channel
in proliferation. The loss of expression of the two
channels KV 1.1 and KV 1.3 may correspond to their role
in apoptosis