Improved perfusion conditions for patch-clamp recordings on human erythrocytes.
Various configurations of the patch-clamp method are powerful tools for examining the transport of charged solutes across biological membranes. Originally developed for the study of relatively large cells which adhere to solid surfaces under in vitro culture, these methods have been increasingly applied to small cells or organelles in suspension. Under these conditions, a number of significant technical problems may arise as a result of the smaller geometry. Here, we examined these problems using human erythrocytes infected with the malaria parasite, Plasmodium falciparum, a system where experimental differences and the technical difficulty of erythrocyte patch-clamp have hindered universal agreement on the properties of the induced ion channels. We found that patch-clamp recordings on infected erythrocytes are especially susceptible to artifacts from mechanical perturbations due to solution flow around the cell. To minimize these artifacts, we designed a new perfusion chamber whose geometry allows controlled solution flow around the fragile erythrocyte. Not only were recordings acquired in this chamber significantly less susceptible to perfusion artifacts, but the chamber permitted rapid and reversible application of known inhibitors with negligible mechanical agitation. Electrophysiological recordings then faithfully reproduced several findings made with more traditional methods. The new perfusion chamber should also be useful for patch-clamp recordings on blood cells, protoplasts, and organelles.
Lisk
Godfrey
G
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Desai
Sanjay A
SA
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004912:Erythrocytes / Q000469:parasitology* / Q000502:physiology*; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods; D010477:Perfusion / Q000295:instrumentation* / Q000379:methods; D010963:Plasmodium falciparum / Q000502:physiology*