Supplementary Figure 5: Cu²⁺-TETAC binding to L186C site does not significantly change the electrophysiological properties of spHCN channels.

a-d, Left: representative current traces of spHCN-L186C channels with L-Anap incorporated at S346, L348, S353, and W355 sites of the S4 helix before and after applying 10 µM Cu²⁺-TETAC. Currents were recorded in the inside-out patch configuration and elicited by a series of hyperpolarizing voltage pulses from 0 mV to -110 mV (-100 mV for S353) with 10 mV steps, in the presence of 1 mM cAMP. Right: Normalized G-V relationships for the same recordings on the left before and after applying 10 µM Cu²⁺-TETAC. e, Time course of the Anap fluorescence for spHCN-W355Anap, L186C channels before and during application of 10 µM Cu²⁺-TETAC, illustrating that Anap fluorescence was not further decreased after 2 mins of applying Cu²⁺-TETAC compared to 1 min. This suggests the labeling of L186C was complete within 1 minute of Cu²⁺-TETAC application. n = 3, error bars are s.e.m.