MEMBRANE-TRANSPORT MECHANISMS PROBED BY CAPACITANCE MEASUREMENTS WITHMEGAHERTZ VOLTAGE-CLAMP

We have used capacitance measurements with a 1-mu s voltage clamp tech nique to probe electrogenic ion-transporter interactions in giant exci sed membrane patches, The hydrophobic ion dipicrylamine was used to te st model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capac itance, monitored by 1-mV sinusoidal perturbations, correspond to sing le charges moving across 76% of the membrane field at a rate of 9500 s (-1) at 0 mV. For the cardiac Na,K pump, the combined presence of cyto plasmic ATP-and sodium induces an increase of apparent membrane capaci tance which requires the presence of extracellular sodium, The depende ncies of capacitance changes on frequency, voltage, ATP, and sodium ve rify that phosphorylation enables a slow 300- to 900-s(-1), pump trans ition (the E(1)-E(2) conformational change), which in turn enables fas t, electrogenic, extracellular sodium binding reactions, For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oo cyte membrane, we find that chloride binding from the cytoplasmic side , and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturb ation frequencies. Evidently, ion binding by the GAT1 transporter supp resses an intrinsic fast charge movement which mag originate from a mo bility of charged residues of the transporter binding sites. The resul ts demonstrate that fast capacitance measurements can provide new insi ght into electrogenic processes closely associated with ion binding by membrane transporters.