Cu²⁺ affects gating of Ca_v2.3 through the extracellular binding site in a state-dependent manner. (A) Time dependence of the inhibitory effect of 13 nM Cu²⁺ on currents through Ca_v2.3 channels, induced by depolarization to −20 or 20 mV (a). The percentage of inhibition at each time point was calculated as a difference in current amplitudes measured in Tricine (black traces) and Cu²⁺ solutions (gray traces), divided by the amplitude in Tricine (b). (B) Voltage dependence of the kinetics of facilitation of Cu²⁺ inhibition. Time constants were determined from A by fitting time dependences with the monoexponential functions (n = 6). (C) Voltage dependence of the inhibition of current through Ca_v2.3 channels by 13 nM Cu²⁺. The percentage of inhibition at each voltage was calculated as a difference between peak current amplitudes measured in Tricine and Cu²⁺ solutions divided by the current amplitude in Tricine (n = 6). (D) Strong depolarization partially reverses the inhibitory effect of Cu²⁺ on Ca_v2.3. Current traces were recorded in response to the protocol depicted above in Tricine- (a) and Cu²⁺-containing (b) solutions (13 nM). (E) Quantification of the Cu²⁺ inhibition of Ca_v2.3 current, recorded before ((T₁−C₁)/T₁) or after ((T₂−C₂)/T₂) the delivery of a depolarizing pulse to 150 mV (n = 5; ***, P < 0.0001 with paired Student’s t test).