Figure 3.

Effects of Ca²⁺ and CaM mutant on Trp3 activity in inside-out patches. (A–C) Single-channel activities in patches excised from Trp3-expressing HEK 293 cells to a Ca²⁺-free solution. (A) Representative traces recorded from a patch at a transmembrane potential of +60 or −60 mV. (B and C) Amplitude (B) and open time (C) histograms generated from idealized traces encompassing 90-s recordings. Note the actual open time is very short for Trp3 (<0.2 ms) (18), and thus there are many incompletely resolved events in the records. Because of this, the mean amplitudes obtained from Gaussian fits are underestimations, and the mean open times obtained by fitting with one exponential are overestimations of the real values. (D) Effect of Ca²⁺ on the basal Trp3 single-channel activity in excised inside-out patches. A patch held at −60 mV was perfused sequentially with intracellular solution containing 5 mM EGTA and no added Ca²⁺ (0 Ca²⁺) and then a solution containing 1.8 μM Ca²⁺. Current recovered in 0 Ca²⁺. Time plots show mean currents under each condition in 0.4-s bins. Representative traces are shown above the plots. Dashed lines indicate zero current levels. (E) Averages ± SEM (n = 11) of inhibition of Trp3 basal activity by Ca²⁺. Relative activity was calculated based on NP_o, which is defined as mean current/unitary current amplitude and was calculated from idealized traces with the use of pclamp8. (F) Expression of a mutant (Upper) but not wild-type (Lower) CaM increased Trp3 basal activity in excised patches. For cells overexpressing the wild-type CaM, there was no activity under both conditions and therefore the time plots show baseline fluctuations. (G) Average ± SEM of mean currents for numbers of patches shown in parentheses. P < 0.01 different from Trp3 alone (*) or from 0 Ca²⁺ (**) by Student's t test.