A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance

Abstract

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug-protein interactions involving this mutant residue.

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

“…Furthermore, reversal mutations in these alleles at residue 76 that were previously shown to be critical to CQ resistance in Plasmodium itself [10], mirror their functional effects on lysosomal pH in this heterologous expression system.…”

“…The amino acid residue at position 76 is directly correlated with CQ resistance in the parasite itself [3,10]. In CQS patient isolates a lysine residue is present at this position, whereas, in CQR patient isolates the lysine is replaced by threonine [11].…”

“…Numerous studies have implicated the PfCRT residue at position 76 as being key to CQ resistance [3,10]. CQR patient isolates have a threonine at this position whereas CQS isolates have a lysine.…”

“…CQ-resistant (CQR) patient isolates contain a K76T mutation in a background that contains several other mutations [5]. Expression of the "back mutation" of T76K in a CQ-resistant allele increases the CQ susceptibility [10]. The endogenous function of PfCRT and the mechanism by which it confers CQ resistance is a subject of intense investigation [see 11, for review].…”

“…For example, while verapamil, a calcium channel antagonist, reverses CQ resistance [21], the mechanism of reversal is uncertain, despite evidence that PfCRT is involved [5,10,20]. Physiological and biochemical studies of Plasmodium proteins in situ are time consuming and subject to technical barriers [22].…”