Fig. 5.

Inactivation of human norepinephrine transporter (hNET)-myc and I155C hNET mixtures by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). HeLa cells were transfected with mixtures of hNET-myc and I155C, as described in the legend to Fig. 3, and were assayed for dopamine (DA) transport. The expression of I155C relative to total hNET was determined by quantitative immunoblotting and is indicated on the x-axis. The normalized mean of three separate experiments is shown, with error bars indicating SD. Transport rates before (●) and after (◯) treatment with 2.5 mM MTSET for 10 min. The solid line is the best fit to the control transport rates. The shortdashed line is the activity expected if no interaction occurred between resistant and sensitive forms and the amount of inactivation was equal to the amount of activity contributed by I155C. The long-dashed line represents the calculated activity if hNET was a dimer and modification of both subunits was required for inactivation of activity in that dimer. The dot-dashed line represents the calculated activity if modification of one subunit in a dimer inactivated all the activity of that dimer. All calculations assumed random dimer formation. Mean nonspecific uptake was determined in HeLa cells transfected with the parent vector pBluescript SK II(–) alone and was subtracted from total uptake to yield specific DA uptake.