Substrate Binding to Serotonin Transporters Reduces Membrane Capacitance

Abstract

Monoaminergic signal transmission is terminated by reuptake of monoamines via the closely related transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). SERT is a secondary active transporter that harnesses the chemical energy stored in the gradients of Na+, Cl- and K+ to transport 5-HT uphill against an opposing concentration gradient.The transport cycle of SERT is complex and encompasses several partial-reactions, some of which can directly be monitored utilizing measurements of currents mediated by SERT. However, these measurements are limited to ionic conditions that support currents but fail to provide insights at conditions at which SERT is electrically silent.We therefore performed capacitance measurements during rapid serotonin application and observed that substrate binding to SERT decreases the membrane capacitance. This effect was absent in control cells.Our analysis suggests that the reduction in membrane capacitance occurs as a consequence of the adsorption of 5-HT to a charged residue in the binding site of SERT. This process eliminates negative extracellular surface charges. Surface charge elimination is accompanied by a change in the surface potential, concomitantly affecting the transmembrane potential. The change of the latter then gives rise to a change in the measured membrane capacitance. The substrate-induced decrease in membrane capacitance was still present, when omitting co-substrates. This is consistent with our hypothesis that the reduced capacitance resulted from serotonin binding (adsorption). At sodium-free conditions, the affinity for serotonin was decreased which was reflected by a smaller capacitance reduction. Our findings suggest random order binding reactions of substrate and co-substrates and that serotonin and Na+ bind in a cooperative manner.Financial support was given to WS by the Austrian Funds FWF (P28090-B27).