Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes.
Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.
Department of Physiology and Universitätsklinik für Anaesthesiologie und Transfusionsmedizin, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Philipp A
PA
Lang
Stephanie
S
Kaiser
Swetlana
S
Myssina
Christina
C
Birka
Christof
C
Weinstock
Hinnak
H
Northoff
Thomas
T
Wieder
Florian
F
Lang
Stephan M
SM
Huber
D017302:Annexins / Q000378:metabolism; D017209:Apoptosis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D003022:Clotrimazole / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005434:Flow Cytometry; D006460:Hemolysin Proteins / Q000633:toxicity*; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000502:physiology; D014736:Vibrio parahaemolyticus
D017302:Annexins; D002412:Cations; D006460:Hemolysin Proteins; D015221:Potassium Channels; D015759:Ionomycin; D003022:Clotrimazole; D011188:Potassium; D002118:Calcium