Figure 6.

Surface-expression and steady-state activation characteristics of rSlo were not affected by the coexpression of rANKRA. (A) Effects of rANKRA on the surface expression of rSlo channel. Cell lysates containing 60 μg of proteins and biotinylated proteins from equal amount of cell lysates were loaded and monitored using α-hSlo antibody (left). Quantification of membrane surface expression of rSlo. The biotinylated fractions of rSlo proteins were quantified by dividing their densitometric intensities of rSlo bands by those of total lysates. Each value represents the mean ± SE of three experiments from different cell batches (right). None of value differs from each other at the p < 0.05 level (paired Student's t test). (B) Representative macroscopic currents of rSlo::EGFP and rSlo::EGFP/rANKRA recorded in symmetrical 120 mM K⁺ solutions containing free [Ca²⁺]_i of 0.1, 1.0, 2.0, and 20.0 μM, as indicated. For ionic currents, the potential was held at 0 mV, stepped to test potential ranging from –140 to +140 mV in 10-mV increments, and then stepped down to –100 mV to measure tail currents. Each current traces represent an average of three records in succession. (C) G-V relationships for rSlo::EGFP (open symbols) and rSlo::EGFP/rANKRA (closed symbols) were not changed significantly. Each symbol represents the value obtained from four different [Ca²⁺]_i; 0.1 μM (square), 1.0 μM (circle), 2.0 μM (triangle), and 20.0 μM (diamond). Conductances (G) were measured at tail currents 1 ms after a tail step pulse and normalized by observed maximum conductances (G_max). Data points were plotted the mean from four different patches for rSlo::EGFP or six for rSlo::EGFP/rANKRA. The lines in G-V represents the best fit of data points to Boltzmann equation G = G_max/{1 + exp[(–V + V₅₀) x zF/RT]}, where V₅₀ is the half-activation voltage, z is the gating valence, F is Faraday's constant, R is the gas constant, and T is temperature (rSlo::EGFP, dotted line; rSlo::EGFP/rANKRA, solid line). (D) Calcium-dependent activation characteristics were not altered by the expression of rANKRA. Ca²⁺-dependent activation of rSlo::EGFP with or without rANKRA protein measured at +80 mV. Tail currents normalized by the maximum response evoked by 20 μM Ca²⁺ were plotted as a function of intracellular calcium concentration, and the data points were fitted with Hill equation, I = I_max ·[Ca ²⁺] _iⁿ/(K_d ^App + [Ca²⁺]_iⁿ). Each data point represents an average of several independent data sets (rSlo::EGFP, N = 4 and rSlo::EGFP/rANKRA, N = 5); error bars indicate SEM.