ABSTRACT
We describe the expression of gpIRK1, an inwardly rectifying K+ channel obtained from guinea pig cardiac cDNA. gpIRK1 is a homologue of the mouse IRK1 channel identified in macrophage cells. Expression of gpIRK1 in Xenopus oocytes produces inwardly rectifying K+ current, similar to the cardiac inward rectifier current IK1. This current is blocked by external Ba2+ and Cs+. Plasmids containing the gpIRK1 coding region under the transcriptional control of constitutive (PGK) or inducible (GAL) promoters were constructed for expression in Saccharomyces cerevisiae. Several observations suggest that gpIRK1 forms functional ion channels when expressed in yeast. gpIRK1 complements a trk1 delta trk2 delta strain, which is defective in potassium uptake. Expression of gpIRK1 in this mutant restores growth on low potassium media. Growth dependent on gpIRK1 is inhibited by external Cs+. The strain expressing gpIRK1 provides a versatile genetic system for studying the assembly and composition of inwardly rectifying K+ channels.
Subject(s)
Potassium Channels, Inwardly Rectifying , Potassium/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Base Sequence , Biological Transport/drug effects , Cesium/pharmacology , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression Regulation, Fungal , Genetic Complementation Test , Guinea Pigs , Molecular Sequence Data , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocardium/chemistry , Potassium Channels , Promoter Regions, Genetic , Sequence Alignment , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.
