CHLOROQUINE-RESISTANT ISOFORMS OF THE Plasmodium falciparum CHLOROQUINE RESISTANCE TRANSPORTER ACIDIFY LYSOSOMAL pH IN HEK293 CELLS MORE THAN CHLOROQUINE-SENSITIVE ISOFORMS*
| Autor: | David C. Reeves, Paul D. Roepe, David A. Liebelt, David A. Fidock, Viswanathan Lakshmanan, Myles H. Akabas |
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| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: |
Plasmodium falciparum
Drug Resistance Protozoan Proteins Vacuole Drug resistance Protein Sorting Signals medicine.disease_cause Transfection Article Cell Line Antimalarials Chloroquine Lysosome parasitic diseases medicine Animals Humans Protein Isoforms Molecular Biology Mutation biology Membrane transport protein Membrane Proteins Membrane Transport Proteins Hydrogen-Ion Concentration biology.organism_classification Virology Molecular biology Protein Structure Tertiary medicine.anatomical_structure biology.protein Parasitology Calcium Heterologous expression Lysosomes medicine.drug |
| Popis: | 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 Ca2+ 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. |
| Databáze: | OpenAIRE |
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