Figure 9.

The TRPC5 open channel I-V relationship does not change during intracellular calcium potentiation. (A) CCh-activated TRPC5 currents were elicited by 15-ms voltage steps to +100 mV, followed by 40-ms steps to multiple hyperpolarizing potentials (applied at 1 Hz). The black trace shows the current recorded ∼15 s after the addition of 100 µM CCh, and the red line shows the peak currents recorded 91 s after CCh addition (lowercase letters indicate sweep timing in B). (B) The average CCh-activated TRPC5 currents at +100 mV (triangles, final 3 ms of first +100-mV conditioning pulse) and −40 mV (squares, 10-ms average at sweep onset) during the application of 2 Ca external (open bars) and 2 Ba external solution (black bar). a, the timing of sweeps used for subtraction of the uncompensated capacity current. (C) Capacity-corrected traces from A were normalized to the amplitude of the TRPC5-mediated current 0.7 ms after repolarization to +60 mV to account for the large difference in absolute current amplitudes. The smaller relative +100-mV current in c likely indicates that the open probability at +60 and +100 mV are more similar after potentiation. The resulting instantaneous tail currents were similar. (Inset) The tail current at −60 mV on an expanded scale. The solid red lines show single-exponential fits to the current. (D) The size of the CCh-activated TRPC5 tail currents after normalization (as in C) was measured in three to six cells both before (black open circles, ∼30 or fewer seconds after CCh addition) and after (red squares, 100 s or fewer after CCh, when current reached peak amplitude) potentiation. Currents were measured 0.7–0.85 ms after the +100-mV conditioning pulse to allow any uncorrected fast capacity current to settle. Error bars show SEM; absent error bars are smaller than symbol. Internal solution (in mM): 150 Cs-Asp, 2 MgCl₂, 0.36 CaCl₂, 1 EGTA, 4 MgATP, 0.3 NaGTP, and 10 HEPES, pH 7.20 with CsOH (100 nM calculated free [Ca²⁺]). External solution: 2 Ca external and 2 Ba external (CaCl₂ completely replaced by BaCl₂; all else the same).