Voltage Clamp Fluorometry for S4 Movement

For voltage clamp fluorometry (VCF) experiments, a single cysteine was introduced in the extracellular side of S4 of spHCN channels. S4 movement in Shaker K channels was measured by introducing a cysteine at position 350 and labeling this cysteine with Alexa-488-maleimide. Oocytes expressing spHCN channels were incubated for 30 min with 100 µM Alexa-488 C5-maleimide (Molecular Probes). After washout, fluorescent-labeled oocytes were placed animal-pole “up” in a bath housed on a Leica DMLFS upright fluorescence microscope. The light was focused on the animal pole of the oocyte through a 20x objective and was passed through a filter cube from Chroma 41,026 (HQ495/30x, Q515LP, HQ545/50 m). Using VCF, the current and the fluorescence were recorded simultaneously. Fluorescence signals were low-pass Bessel filtered (Frequency Devices) at 200 Hz and digitized at 1 kHz. VCF has previously been shown to detect S4 movement in both Shaker K channels and spHCN channels [30 (link),31 (link),43 (link),49 (link),50 (link),51 (link)]. For cadmium experiments, 50 nL of 10 µM CdCl₂ was injected into voltage-clamped oocytes using a Drummond “Nanoject II” nanoliter injector (Drummond Scientific Co., Broomall, PA, USA).