RESUMO
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.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Potássio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico/efeitos dos fármacos , Césio/farmacologia , Clonagem Molecular , DNA Complementar/genética , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Cobaias , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/química , Canais de Potássio , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
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.
