Isolation and Measurement of CaV2.2 Currents

We used pClamp version 10 software and the Axopatch 200A (Molecular Devices) for data acquisition; data were filtered at 2 kHz (–3 dB) and sampled at 20 kHz. We compensated series resistance by 70–90% with a 7-μs lag and performed online leak correction with a P/–4 protocol. All recordings were obtained at RT. To measure current amplitudes, we either isolated the Ca_V2.2 component by ω-conotoxin GVIA (2 μM) subtraction or we used a combination of inhibitors of Ca_V1 and Ca_V2.1 currents with a mixture of isradipine (10 μM) and ω-agatoxin IVA (50 nM). For the ω-conotoxin GVIA (2 μM) subtraction method, we first recorded the whole-cell Ca_V current, inhibited the Ca_V2.2 component, and subtracted the resistant current (non-Ca_V2.2) from the whole-cell Ca_V current to isolate the Ca_V2.2 current (Andrade et al., 2010 (link)). All cells were maintained in a NaCl external solution (135 mM NaCl, 4 mM MgCl₂, 10 mM CaCl₂, and 10 mM HEPES, pH 7.2 w/NaCl-OH) and, after whole-cell formation, cells were lifted and placed in the stream of a TEA-Cl based solution applied via a microperfusion system (VC-M6, Warner Instruments).