Dose responses for tolbutamide inhibition of channels obtained by coexpression of free SUR1, SUR2A, and Kir6.2 suggest the coassembly of free SUR1 with free SUR2A. (A) Representative current traces obtained in inside-out patches from oocytes expressing SUR1+Kir6.2 (black trace), SUR2A+Kir6.2 (blue trace), or SUR1+SUR2A+Kir6.2 (SUR1:SUR2A=1:1; red trace). The membrane was held at −100 mV and tolbutamide at various concentrations was applied to the bath solution as indicated. The green line indicates the application of 2.5 mM ATP. The biphasic current increase during the washout of ATP is a perfusion artifact. (B) Fractional currents remaining in the presence of various concentrations of cytosolic tolbutamide in patches excised from oocytes expressing SUR1+Kir6.2 (black symbols), SUR2A+Kir6.2 (blue symbols), or SUR1+SUR2A+Kir6.2 (red symbols). The solid black line is a fit of Eq. 1 to the SUR1+Kir6.2 data, yielding fit parameters K_i1 = 14 ± 4 μM, h₁ = 0.83 ± 0.15, K_i2 = 2.4 ± 0.2 mM, h₂ = 2.8 ± 0.9, and L=0.51 ± 0.04. The blue solid line is a fit to the Hill equation of the data for SUR2A+Kir6.2, yielding fit parameters K_i = 7.19 ± 0.06 mM, n = 1.74 ± 0.02. Red dotted and solid lines illustrate predicted curves for SUR1+SUR2A+Kir6.2 assuming that SUR1 does not coassemble with SUR2A. The red dotted line is the simple arithmetic average of the two fit functions obtained for SUR1+Kir6.2 (black line) and SUR2A+Kir6.2 (blue line), expected if SUR1 and SUR2A protein was produced at an equal molar ratio but did not coassemble. The red solid line is the best fit to the SUR1+SUR2A+Kir6.2 data by a mixture of the two parent functions, with the fractional amplitude of channels formed from SUR1+Kir6.2, a₁, left as a free parameter. This fit returned a₁ = 0.81 ± 0.07, but still failed to describe the data.