Electrophysiological characterization of the polyspecific organic cation transporter plasma membrane monoamine transporter

Abstract

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation (OC) transporter that transports a variety of endogenous biogenic amines and xenobiotic cations. Previous radiotracer uptake studies showed that PMAT-mediated OC transport is sensitive to changes in membrane potential and extracellular pH, but the precise role of membrane potential and protons on PMAT-mediated OC transport is unknown. Here, we characterized the electrophysiological properties of PMAT in Xenopus laevis oocytes using a two-microelectrode voltage-clamp approach. PMAT-mediated histamine uptake is associated with inward currents under voltage-clamp conditions, and the currents increased in magnitude as the holding membrane potential became more negative. A similar effect was also observed for another cation, nicotine. Substrate-induced currents were largely independent of Na+ but showed strong dependence on membrane potential and pH of the perfusate. Detailed kinetic analysis of histamine uptake revealed that the energizing effect of membrane potentials on PMAT transport is mainly due to an augmentation of Imax with little effect on K0.5. At most holding membrane potentials, Imax at pH 6.0 is approximately 3- to 4-fold higher than that at pH 7.5, whereas K0.5 is not dependent on pH. Together, these data unequivocally demonstrate PMAT as an electrogenic transporter and establish the physiological inside-negative membrane potential as a driving force for PMAT-mediated OC transport. The important role of membrane potential and pH in modulating the transport activity of PMAT toward OCs suggests that the in vivo activity of PMAT could be regulated by pathophysiological processes that alter physiological pH or membrane potential.

Fig. 1.

Inward currents induced by 2.5 mM histamine at pH 6 and 7.5 in the presence (NaCl) or absence (NMDG chloride) of Na⁺. There were no detectable currents when water-injected oocytes were exposed to histamine under similar experimental conditions.

Fig. 2.

Influence of pH on histamine-induced currents in the presence of Na⁺. PMAT-expressing oocytes were exposed to histamine (2.5 mM) in a NaCl-containing buffer at different pH. The data show that histamine-induced currents increased when the pH was made acidic.

Fig. 3.

a, I-V relationship for histamine-induced currents at different experimental conditions. Inward currents induced by 2.5 mM histamine were monitored in PMAT-expressing oocytes at different testing membrane potentials. The data show that H⁺ enhanced the currents, both in the presence (NaCl) and absence (NMDG chloride) of Na⁺, and that hyperpolarization also enhanced the currents, indicating the role of membrane potential in the energization of the transporter. b, I-V relationship for nicotine-induced currents in Na⁺-containing or Na⁺-free perfusates at pH 7.5. Inward currents induced by 2.5 mM nicotine were monitored in PMAT-expressing oocytes at different testing membrane potentials. The data show that hyperpolarization enhanced the currents.

Fig. 4.

Influence of membrane potential on I_max (a) and K_0.5 (b) for histamine-induced currents. PMAT-expressing oocytes were exposed to increasing concentrations of histamine at varying testing membrane potentials over the range of −10 to −150 mV. The substrate-dependent currents were subjected to kinetic analysis according to the Michaelis-Menten equation, and the kinetic parameters, I_max and K_0.5, were calculated. The data show that hyperpolarization increases the magnitude of the maximal currents induced by histamine. This effect is apparent at pH 7.5 and 6, both in the presence (NaCl) and absence (NMDG chloride) of Na⁺. The data also show that hyperpolarization has very little effect on the affinity of the transporter for histamine.

Fig. 5.

Uptake of radiolabeled histamine in PMAT-expressing oocytes. Uptake of radiolabeled histamine (100 μM) was measured in water-injected and PMAT cRNA-injected oocytes at pH 6 in the presence (NaCl) or absence (NMDG chloride) of Na⁺. *, significantly (p < 0.0001) different from water-injected oocytes.