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Human TRPC6 expressed in HEK 293 cells forms non‐selective cation channels with limited Ca 2+ permeability
Author(s) -
Estacion Mark,
Sinkins William G.,
Jones Stephen W.,
Applegate Milana A. B.,
Schilling William P.
Publication year - 2006
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2005.103143
Subject(s) - trpc6 , depolarization , extracellular , chemistry , biophysics , phospholipase c , hek 293 cells , cytosol , membrane potential , diacylglycerol kinase , transient receptor potential channel , biochemistry , protein kinase c , receptor , biology , signal transduction , enzyme
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 m m . 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 (∼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 n m 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.