Dmlfs upright fluorescence microscope

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).

Oocytes were injected with 9.2 ng of cRNA encoding of psWT K_v7.1 (C214A/G219C/C331A) alone or simultaneously with 2.3 ng of cRNA encoding wt CiVSP. After incubation for 3–7 days at 18 C, the cells were labeled in high K⁺ labeling solution (98 mmol L⁻¹ KCl, 1.8 mmol L⁻1 CaCl2, 5 mmol L⁻¹ HEPES, pH 7.6) with 10 μM Alexa 488 C₅-maleimide (Molecular Probes) for 45 min on ice, washed with ND96 solution (96 mmol L⁻¹ NaCl, 4 mmol L⁻¹ KCl, 1.8 mmol L⁻¹ CaCl₂, 1 mmol L⁻¹ MgCl₂, 5 mmol L⁻¹ HEPES), and kept on ice until recording to minimize recycling of labeled channels. VCF recordings were carried out in ND96 solutions + /- (R)-L3. Fluorescence recordings were performed using a Leica DMLFS upright fluorescence microscope with a FITC filter cube. Emission from the animal pole was focused onto a pin 20 A photodiode (OSI Optoelectronics) and amplified using an EPC10 (HEKA) patch amplifier and analog filtered at 200 Hz. Simultaneous two-electrode voltage-clamp recordings were measured using a Dagan CA1-B amplifier in TEVC mode. Current and fluorescence signals were digitized at 1 kHz.