title: Human TRPC6 expressed in HEK 293 cells forms non-selective cation channels with limited Ca2+ permeability.
abstract: TRPC6 is thought to be a Ca(2+)-permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) when the cells were bathed in high-K(+) buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca(2+) permeability relative to monovalent cation permeability and/or that Ca(2+) influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca(2+) permeability, TRPC6 currents were examined in extracellular buffer in which Ca(2+) was varied from 0.02 to 20 mm. The results were consistent with a pore-permeation model in which Ca(2+) acts primarily as a blocking ion and contributes only a small percentage ( approximately 4%) to whole-cell currents in the presence of extracellular Na(+). Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca(2+)](i) 50-100 nm when the membrane potential was clamped at between -50 and -80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca(2+)-activated K(+) current), activation of TRPC6 will lead to a sustained increase in [Ca(2+)](i). Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca(2+) response will be cell specific and dependent upon the complement of other channel types.
authors.affiliation: Rammelkamp Center for Education and Research, MetroHealth Medical Center, Cleveland, OH 44109-1998, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Estacion
authors.forename: William G
authors.initials: WG
authors.lastname: Sinkins
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Jones
authors.forename: Milana A B
authors.initials: MA
authors.lastname: Applegate
authors.forename: William P
authors.initials: WP
authors.lastname: Schilling
mesh_terms: D002118:Calcium / Q000493:pharmacokinetics* / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D003512:Cyclohexanones / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004594:Electrophysiology; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000737:chemistry* / Q000166:cytology / Q000196:embryology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D012964:Sodium / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000032:analysis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D000074287:TRPC6 Cation Channel
chemical_list: D003512:Cyclohexanones; D004075:Diglycerides; D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011480:Protease Inhibitors; D011760:Pyrrolidinones; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; C038237:1-oleoyl-2-acetylglycerol; D012964:Sodium; D011188:Potassium; D002118:Calcium