Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes.

The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes.

Diverse mutational pathways converge on saturable chloroquine transport via the malaria parasite's chloroquine resistance transporter.

Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.

Multiple drugs compete for transport via the Plasmodium falciparum chloroquine resistance transporter at distinct but interdependent sites.

Mutations in the "chloroquine resistance transporter" (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.

Organization of rat vibrissa motor cortex and adjacent areas according to cytoarchitectonics, microstimulation, and intracellular stimulation of identified cells.

The relationship between motor maps and cytoarchitectonic subdivisions in rat frontal cortex is not well understood. We use cytoarchitectonic analysis of microstimulation sites and intracellular stimulation of identified cells to develop a cell-based partitioning scheme of rat vibrissa motor cortex and adjacent areas. The results suggest that rat primary motor cortex (M1) is composed of three cytoarchitectonic areas, the agranular medial field (AGm), the agranular lateral field (AG1), and the cingulate area 1 (Cg1), each of which represents movements of different body parts. Vibrissa motor cortex corresponds entirely and for the most part exclusively to AGm. In area AG1 body/head movements can be evoked. In posterior area Cg1 periocular/eye movements and in anterior area Cg1 nose movements can be evoked. In all of these areas stimulation thresholds are very low, and together they form a complete representation of the rat's body surface. A strong myelinization and an expanded layer 5 characterize area AGm. We suggest that both the strong myelinization and the expanded layer 5 of area AGm may represent cytoarchitectonic specializations related to control of high-speed whisking behavior.