Human TRPC6 expressed in HEK 293 cells forms non‐selective cation channels with limited Ca<sup>2+</sup> permeability

Estacion, Mark; Sinkins, William G.; Jones, Stephen; Applegate, Milana A. B.; Schilling, William P.

doi:10.1113/jphysiol.2005.103143

Cited by 111 publications

(95 citation statements)

References 41 publications

(65 reference statements)

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“…These data are fully consistent with a TRPC6 permeation model in which Ca 2ϩ contributes only a small percentage (Ϸ4%) to whole-cell currents in the presence of extracellular Na ϩ (29). The resulting Na ϩ influx through TRPC6 channels then leads to membrane depolarization and activation of voltage-gated calcium channels (29)(30)(31). Moreover, inhibition of potassium channels as a secondary event may also contribute to depolarization, because an elevation of intracellular Na ϩ is able to block potassium channels (32).…”

Section: Isolation Of Pasmc and Expression Of Trpc Subtypes In Pasmcsupporting

confidence: 74%

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Weißmann

Dietrich

Fuchs

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

271

285

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Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6 ؊/؊ mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6 ؊/؊ but not in WT mice. This effect was mirrored by a lack of hypoxiainduced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6 ؊/؊ mice. In both WT and TRPC6 ؊/؊ PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6 ؊/؊ PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.hypoxia-induced diacylglycerol accumulation ͉ precapillary pulmonary arterial smooth-muscle cells ͉ pulmonary hypertension ͉ transient receptor potential channel 6-deficient mouse model ͉ arterial hypoxemia A cute regional hypoxic pulmonary vasoconstriction (HPV) is necessary to maintain optimized gas exchange by directing blood flow from poorly ventilated to well ventilated areas of the lung. Under conditions of generalized hypoxia, however, total pulmonary vascular resistance rises with subsequent increase of right heart load (1-3). Chronic hypoxia, as occurring in ventilatory disorders induces chronic pulmonary hypertension, pulmonary vascular remodeling, and cor pulmonale (4). The underlying oxygen sensing and signal transduction mechanisms of the acute and chronic vascular responses are largely unknown. A rise of intracellular calcium ([Ca 2ϩ ] i ) in pulmonary artery smooth-muscle cells (SMCs) has been suggested to be the key event in these processes (5-8). However, the question how [Ca 2ϩ ] i is regulated has not yet been resolved. Among others, transient receptor potential (TRP) channels are regulators of [Ca 2ϩ ] i . The TRP protein superfamily consists of a diverse group of nonselective cation channels involved in many basic cellular processes (9). Whereas members of the TRPV and TRPM subfamilies have emerged as versatile cellular sensors, the functional importance of the seven members (TRPC1 to -7) of the TRPC (transient receptor potential cation channel subfamily C) subfamily...

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Section: Isolation Of Pasmc and Expression Of Trpc Subtypes In Pasmcsupporting

confidence: 74%

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Weißmann

Dietrich

Fuchs

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

271

285

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“…Specifically, TRPC6 behaves as a nonselective cation channel that is activated by diacylglycerol in a membranedelimited fashion, independently of protein kinase C (37). The TRPC6 channel is not considered to be store-operated and only has limited Ca 2+ permeability relative to monovalent cation permeability (38). We cannot rule out their contributions at this time, but our results indicate that TRPV6 has a major influence on cellular calcium entry.…”

Section: Discussionmentioning

confidence: 46%

The role of TRPV6 in breast carcinogenesis

Bolanz

Hediger

Landowski

2008

Molecular Cancer Therapeutics

185

163

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TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue. In this study, we further confirmed this observation by analysis of breast cancer tissues, which indicated that TRPV6 mRNA expression was up-regulated between 2-fold and 15-fold compared with the average in normal breast tissue. Whereas TRPV6 is expressed in the cancer tissue, its role as a calcium channel in breast carcinogenesis is poorly understood. Therefore, we investigated how TRPV6 affects the viability, apoptosis, and calcium transport in the breast cancer cell line T47D. Hormones can also affect the tumor development; hence, we determined the effects of estradiol, progesterone, and 1,25-vitamin D on TRPV6 transcription. Interestingly, the estrogen receptor antagonist tamoxifen reduced expression of TRPV6 and is able to inhibit its calcium transport activity (IC 50 , 7.5 Mmol/L). The in vitro model showed that TRPV6 can be regulated by estrogen, progesterone, tamoxifen, and 1,25-vitamin D and has a large influence on breast cancer cell proliferation. Moreover, the effect of tamoxifen on cell viability was enhanced when TRPV6 expression was silenced with small interfering RNA.

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“…The high Ca 2ϩ reduces amplitudes of inward currents at more negative membrane potentials (12); therefore, current amplitudes measured at ϩ80 mV were used to quantify effectiveness of various treatments. However, for experiments using pressure pulses as a stimulus, the external Ca 2ϩ was reduced to 0.8 mM to facilitate detection of inward currents at Ϫ60 mV (12). Pipette solutions in all experiments contained 10 mM NaCl, 125 mM CsCl, 6.2 mM MgCl 2, 10 mM HEPES, and 10 mM EGTA (pH 7.2).…”

Section: Methodsmentioning

confidence: 99%

Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol

Anderson

Kim

Hagmann

et al. 2013

American Journal of Physiology-Cell Physiology

120

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Anderson M, Kim EY, Hagmann H, Benzing T, Dryer SE. Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol. Am J Physiol Cell Physiol 305: C276 -C289, 2013. First published May 8, 2013 doi:10.1152/ajpcell.00095.2013.-Gain-of-function mutations in the transient receptor potential (TRP) cation channel subfamily C member 6 (TRPC6) gene and mutations in the NPHS2 gene encoding podocin result in nephrotic syndromes. The purpose of this study was to determine the functional significance of biochemical interactions between these proteins. We observed that gating of TRPC6 channels in podocytes is markedly mechanosensitive and can be activated by hyposmotic stretch or indentation of the plasma membrane. Stretch activation of cationic currents was blocked by small interfering RNA knockdown of TRPC6, as well as by SKF-96365 or micromolar La 3ϩ . Stretch activation of podocyte TRPC6 persisted in the presence of inhibitors of phospholipase C (U-73122) and phospholipase A 2 (ONO-RS-082). Robust stretch responses also persisted when recording electrodes contained guanosine 5=-O-(2-thiodiphosphate) at concentrations that completely suppressed responses to ANG II. Stretch responses were enhanced by cytochalasin D but were abolished by the peptide GsMTx4, suggesting that forces are transmitted to the channels through the plasma membrane. Podocin and TRPC6 interact at their respective COOH termini. Knockdown of podocin markedly increased stretch-evoked activation of TRPC6 but nearly abolished TRPC6 activation evoked by a diacylglycerol analog. These data suggest that podocin acts as a switch to determine the preferred mode of TRPC6 activation. They also suggest that podocin deficiencies will result in Ca 2ϩ overload in foot processes, as with gain-of-function mutations in the TRPC6 gene. Finally, they suggest that mechanical activation of TRP family channels and the preferred mode of TRP channel activation may depend on whether members of the stomatin/ prohibitin family of hairpin loop proteins are present.podocyte; glomerular filtration; TRPC6; slit diaphragm; podocyte; mechanosensitive PODOCYTES ARE HIGHLY DIFFERENTIATED multipolar cells that cover the external surface of glomerular capillaries and form an essential component of the kidney ultrafiltration apparatus (46). Primary processes extend from the podocyte cell body and ramify extensively as they wrap around the glomerular capillary. Specialized structures known as foot processes normally emanate at regular spatial intervals from the major processes of podocytes and attach to the surface of the glomerular basement membrane. Connections between interdigitating foot processes, known as slit diaphragms (SDs), are formed by trans-interactions of specialized transmembrane adhesion molecules (26). The interacting extracellular domains of SD proteins form a porous matrix (65) that provides a pathway for convective flow and diffusion of water and small solutes into Bowman's space (7). SDs may have multiple roles, as the...

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Human TRPC6 expressed in HEK 293 cells forms non‐selective cation channels with limited Ca²⁺ permeability

Cited by 111 publications

References 41 publications

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

The role of TRPV6 in breast carcinogenesis

Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol

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Human TRPC6 expressed in HEK 293 cells forms non‐selective cation channels with limited Ca2+ permeability

Cited by 111 publications

References 41 publications

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

The role of TRPV6 in breast carcinogenesis

Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol

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Human TRPC6 expressed in HEK 293 cells forms non‐selective cation channels with limited Ca²⁺ permeability