E3-targeted anti-TRPC5 antibody inhibits store-operated calcium entry in freshly isolated pial arterioles

2006 ◽  
Vol 291 (6) ◽  
pp. H2653-H2659 ◽  
Author(s):  
Shang-Zhong Xu ◽  
Guylain Boulay ◽  
Richard Flemming ◽  
David J. Beech
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Transient Receptor Potential ◽  
Ionic Current ◽  
Receptor Potential ◽  
Muscle Cells ◽  
Calcium Entry ◽  
Store Depletion ◽  
Store Operated Calcium Entry ◽  
Transient Receptor

Smooth muscle cells in arterioles have pivotal roles in the determination of blood pressure and distribution of local blood flow. The cells exhibit calcium entry in response to passive store depletion, but the mechanisms and relevance of this phenomenon are poorly understood. Previously, a role for canonical transient receptor potential 1 (TRPC1) was indicated, but heterologous expression studies showed TRPC1 to have poor function in isolation, suggesting a requirement for additional proteins. Here we test the hypothesis that TRPC5 is such an additional protein, because TRPC5 forms heteromultimeric channels with TRPC1, and RNA encoding TRPC5 is present in arterioles. Recordings were from arteriolar fragments freshly isolated from rabbit pial membrane. Ionic current in response to store depletion has properties like that of the TRPC1/TRPC5 heteromultimer, and so the effect of the E3-targeted, externally acting, anti-TRPC5 blocking antibody (T5E3) was explored. T5E3 suppressed calcium entry in store-depleted arterioles but had no effect in the absence of store depletion. T5E3 preadsorbed to its antigenic peptide did not inhibit calcium entry. TRPC6 is commonly detected in smooth muscle and is present in the arterioles, but T5E3 had no effect on TRPC6. The data suggest that calcium entry occurring in response to passive store depletion in smooth muscle cells of arterioles involves TRPC5 as well as TRPC1.

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

2005 ◽  
Vol 288 (2) ◽  
pp. C245-C252 ◽  
Author(s):  
Shen Zhang ◽  
Jason X.-J. Yuan ◽  
Kim E. Barrett ◽  
Hui Dong
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Muscle Cells ◽  
Reverse Mode ◽  
Store Depletion ◽  
Pulmonary Artery Smooth Muscle ◽  
Transient Receptor

A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca2+ stores opens store-operated Ca2+ channels (SOC) and causes Ca2+ entry. Transient receptor potential (TRP) cation channels that are permeable to Na+ and Ca2+ are believed to form functional SOC. Because sarcolemmal Na+/Ca2+ exchanger has also been implicated in regulating [Ca2+]cyt, this study was designed to test the hypothesis that the Na+/Ca2+ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca2+]cyt by contributing to store depletion-mediated Ca2+ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na+, which switches the Na+/Ca2+ exchanger from the forward (Ca2+ exit) to reverse (Ca2+ entry) mode, significantly increased [Ca2+]cyt, whereas inhibition of the Na+/Ca2+ exchanger with KB-R7943 (10 μM) markedly attenuated the increase in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Store depletion also induced a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Removal of extracellular Na+ or inhibition of the Na+/Ca2+ exchanger with KB-R7943 attenuated the store depletion-mediated Ca2+ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange contributes to store depletion-mediated increase in [Ca2+]cyt, and that blockade of the Na+/Ca2+ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension.

Molecular determinants of PKA-dependent inhibition of TRPC5 channel

2011 ◽  
Vol 301 (4) ◽  
pp. C823-C832 ◽  
Author(s):  
Tae Sik Sung ◽  
Jae Pyo Jeon ◽  
Byung Joo Kim ◽  
Chansik Hong ◽  
Sung Young Kim ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Muscle Cells ◽  
Intestinal Smooth Muscle ◽  
Transient Receptor Potential Vanilloid ◽  
Trpc Channels ◽  
Muscarinic Stimulation ◽  
Transient Receptor

Canonical transient receptor potential (TRPC) channels are Ca2+-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gαs. Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gαs [Gαs (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gαs (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by Gs/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β2-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.

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