Measurement of relative Ca²⁺ permeability during sustained activation of TRPV1 receptors.
Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.
Department of Biology, Clarkson University, 177 Science Center, 8 Clarkson Ave., P.O. Box 5805, Potsdam, NY, 13699-5805, USA. dsamways@clarkson.edu.
Damien S K
DS
Samways
Department of Biology, Clarkson University, 177 Science Center, 8 Clarkson Ave., P.O. Box 5805, Potsdam, NY, 13699-5805, USA.
Evan
E
Tomkiewicz
Department of Biology, Clarkson University, 177 Science Center, 8 Clarkson Ave., P.O. Box 5805, Potsdam, NY, 13699-5805, USA.
Olivia M
OM
Langevin
Department of Biology, Clarkson University, 177 Science Center, 8 Clarkson Ave., P.O. Box 5805, Potsdam, NY, 13699-5805, USA.
Maurish
M
Bukhari
D000200:Action Potentials; D002118:Calcium / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D010539:Permeability; D050916:TRPV Cation Channels / Q000378:metabolism*
D050916:TRPV Cation Channels; D002118:Calcium
Ca2+; Capsaicin; Ion channel; Permeability; TRPV1