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

Abstract

The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification of a Dd2 allele expressing the T76K "back mutation" was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca 2+ stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

“…The resistance to chloroquine observed in P. falciparum is dependent to mutations in specific transporter (PfCRT), by using heterologous expression Reeves et al (2006) observed a increase of lysosomes acidification in mammalian cells expressing PfCRT, providing new information about acidification process and chloroquine resistance. Fig.…”

PfCRT and its role in antimalarial drug resistance

“…A number of heterologous expression studies have raised the possibility that mutations in PfCRT might affect the transport of H + ions across the DV membrane [40, 41, 103, 104]. This is supported by the demonstration that the rate of leakage of H + from the DV (determined by monitoring the rate of DV alkalinization on inhibition of the V-type H + -ATPase with concanamycin A) is greater in pfcrt -modified parasites expressing mutant PfCRT than in an isogenic line expressing wild type PfCRT [39].…”

Intracellular Trafficking and Decondensation Kinetics of Chitosan–pDNA Polyplexes

“…Lysosomal labeling by specific accumulation of fluorescent-dextran following endocytosis has been demonstrated by colocalization with established lysosomal markers such as lysosome-associated membrane protein-2 in many cell types including HEK293. [25][26][27] After 4 hours (Figure 3a,b) of incubation, the intracellular transit of 92-10 polyplexes show a mixture of polyplexes in lysosomes (yellow) and a separate population that is not in lysosomes (red). Subsequently at 12 hours, a stronger colocalization in lysosomes was seen which then diminished at 24-48 hours indicating a gradual loss of colocalization in parallel with a gradual increase of diffuse RITC-chitosan signal in the cell cytoplasm as shown by arrows (Figure 3a).…”