Evidence for Activation of Endogenous Transporters in Xenopus laevis Oocytes Expressing the Plasmodium falciparum Chloroquine Resistance Transporter, PfCRT

Abstract

A large body of genetic, reverse genetic, and epidemiological data has linked chloroquine-resistant malaria to polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum. To investigate the biological function of the chloroquine resistance transporter, PfCRT, as well as its role in chloroquine resistance, we functionally expressed this protein in Xenopus laevis oocytes. Our data show that PfCRT-expressing oocytes exhibit a depolarized resting membrane potential and a higher intracellular pH compared with control oocytes. Pharmacological and electrophysiological studies link the higher intracellular pH to an enhanced amiloride-sensitive H ؉ extrusion and the low membrane potential to an activated nonselective cation conductance. The finding that both properties are independent of each other, together with the fact that they are endogenously present in X. laevis oocytes, supports a model in which PfCRT activates transport systems. Our data suggest that PfCRT plays a role as a direct or indirect activator or modulator of other transporters.

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

“…While the discovery [5] and subsequent study [see for review 11] of the pfcrt gene has added much to our understanding of the molecular mechanism of CQ resistance in malaria, neither the endogenous function, nor the physiological mechanism by which pfcrt confers CQ resistance are clear. Heterologous expression of PfCRT in Pichia pastoris, Saccharomyces cerevisiae [23,24], Dictyostelium discoides [18] and Xenopus laevis [25] has shown that the protein is more amenable to biochemical and physiological analysis in less technically onerous systems than P. falciparum. We chose the HEK293 cell line for heterologous expression, as it is widely used for functional studies of cellular physiology.…”

“…Physiological and biochemical studies of Plasmodium proteins in situ are time consuming and subject to technical barriers [22]. Heterologous expression offers a more convenient system to study PfCRT and to investigate its interactions with drugs [18,[23][24][25]. Here, we demonstrate robust expression of full length PfCRT in human embryonic kidney (HEK293) cells.…”

“…The absence of PfCRT in the plasma membrane was surprising because this is the default location for trafficking of integral membrane proteins, and two previous studies have reported plasma membrane expression of heterologously expressed PfCRT [23,25]. In Xenopus laevis oocytes, the close proximity of the endoplasmic reticulum and Golgi apparatus to the plasma membrane makes it difficult to definitively establish plasma membrane localization using immunofluorescence microscopy on frozen sections [51].…”

Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters

“…The prospect that AeNHE8 is contained in vesicles that fuse with the plasma membrane under conditions of diuresis was ruled out by feeding mosquitoes a blood meal and application of dibutyryl-cAMP to isolated tubules, both of which stimulate Na + excretion but did not alter the localization of the transporter. Efforts to characterize the exchanger that was expressed heterologously in Xenopus laevis oocytes by twoelectrode voltage clamp techniques were frustrated by the activation of well known Na + conductances (Nessler et al, 2004;Reifarth et al, 1999;Tzounopoulos et al, 1995 (Rheault et al, 2007) makes NHAs even more attractive because they could use the high apicalmembrane voltage that is established by the H + V-ATPase (Day et al, 2008).…”

Mutations Conferring Drug Resistance in Malaria Parasite Drug Transporters Pgh1 and PfCRT Do Not Affect Steady-State Vacuolar Ca 2+

“…PfCRT-expressing oocytes exhibit a depolarized membrane potential (⌿ m ) and a higher intracellular pH (pH i ) compared to control oocytes (9). One possibility is that PfCRT interferes with second messengers such as Ca 2ϩ (9).…”