Measurement of relative Ca²⁺ permeability during sustained activation of TRPV1 receptors

Abstract

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 Ca²⁺ entry, and it has been suggested that this channel undergoes a time-dependent change in Ca²⁺ 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 Ca²⁺ current in TRPV1 expressing HEK293 cells show that relative Ca²⁺ 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 Ca²⁺ permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca²⁺ chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca²⁺ are likely due to intracellular accumulation of this ion and a movement of the Ca²⁺ equilibrium potential (E _Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca²⁺ after sustained capsaicin exposure in the absence of added extracellular Ca²⁺, 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 Ca²⁺ permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca²⁺ chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca²⁺ and thus allow for an accurate estimate of this important channel permeability parameter.