Fig. 2. Expression of only pore-forming -subunits produced functional current. (Ai) Family of whole-cell currents from a cell transfected with GFP-Ca_V2.2 evoked by step depolarisations (–60 to +60 mV) from a holding potential of –100 mV. (Aii) Normalised activation curve () was fit by a Boltzmann distribution with a V of +21 mV and a slope of e-fold in 7.8 mV (n=12). By contrast, steady-state inactivation was determined by (prepulse) voltage steps (1-second duration) preceding a test pulse (+30 mV) (n=10). The relationship () was fit by a Boltzmann distribution of V –21 mV. (Bi) Representative macroscopic current sweeps from two cells expressing rSlo₂₇, one dialysed with an electrode solution containing 1 µM free Ca²⁺ (upper) and another dialysed with a solution containing 60 nM free Ca²⁺ (lower). (Bii) Mean normalised current-voltage relationship for cells dialysed with either 1 µM (, n=5) or 60 nM (, n=5) free Ca²⁺. (Ci) Expression of rSlo₂₇ channels was confirmed by western immunoblotting with anti-BK (tsA). Probing the GFP-IP sample from cells co-transfected with rSlo₂₇ and GFP-Ca_V2.2 subunits with anti-BK produced a band of 120 kDa, the predicted molecular mass of the rSlo₂₇ channel -subunit. (Cii) Expression of the GFP-Ca_V2.2 subunit in tsA-201 cell lysates (tsA) was confirmed by western immunoblotting using anti-GFP, with anti-GFP immunoreactivity being absent in the rat whole brain (wb) tissue lane. Immunoreactive bands of 240 and 270 kDa, the predicted molecular masses of the GFP-Ca_V2.2 channel protein, were detected in the GFP-IP by both anti-Ca_V2.2 (lane 1) and anti-GFP (lane 2).