Time-dependent changes in monovalent cation permeability have been reported for P2XR, TRPV1 and TRPA1. An important physiological question is whether relative Ca2+ permeability (PCa/PNa) also changes during prolonged agonist exposure. Previous evidence obtained using current reversal potential (ERev) measurements suggests this to be the case for TRPV1, but it is not known for P2XRs. In our hands, both TRPV1- and P2X-transfected HEK293 cells exhibited time-dependent changes in N-methyl-D-glucamine permeability during prolonged exposure to capsaicin (10 microM) or ATP (30 microM) respectively, which occurred immediately and was complete within 10-15 s. Next we used patchclamp photometry to study time-dependent changes in fractional Ca2+ current (Pf%). These experiments were problematic due to Ca2+ entry often saturating the pipette Ca2+ chelator, fura-2, even under conditions of low external Ca2+. Nevertheless, in experiments where fura-2 saturation did not occur within the first 5-10 seconds of stimulus, the relationship between total charge influx and change in fura-2 fluorescence was linear for both channels, providing no obvious evidence for PCa/PNa changes over this time scale. We considered that saturation of intracellular Ca2+ chelators such as EGTA would likely hinder assessment of time-dependent changes in PCa/PNa using ERev measurements, as these assume that the transmembrane Ca2+ concentration ratio remains constant throughout. In 10 mM external Ca2+, we performed patchclamp photometry using pipettes containing 10 mM fura-2. Cells expressing TRPV1 were stimulated with capsaicin for 25 s, with 140 mV ramps applied every 500 ms. We observed a shift in ERev for the capsaicin-evoked current from 12.2±1.9 to 1.1±1.3 mV (n=4), but this was mostly apparent after the rapid saturation of fura-2 by TRPV1-mediated Ca2+ entry. Therefore, caution is strongly encouraged when interpreting time-dependent changes in Ca2+ permeability based on ERev experiments alone.