Voltage-dependent Ba2+ permeation through store-operated CRAC channels: implications for channel selectivity.
Store-operated Ca2+ entry through CRAC channels is a major route for Ca2+ influx in non-excitable cells. Studies on store-operated channel selectivity using fluorescent dyes have found that the channels are impermeable to Ba2+. Furthermore, in such studies, agonists have been reported to increase Ba2+ influx, leading to the conclusion that additional Ca2+ entry pathways (permeable to Ba2+) co-exist with the Ba2+-impermeable store-operated channels. However, patch clamp experiments demonstrate that CRAC channels are permeable to Ba2+. We have addressed this paradox using fluorescence measurements and whole cell patch clamp recordings of ICRAC. In store-depleted cells loaded with fura 2, Ba2+ application results in a slower and smaller rise in fluorescence than is the case with Ca2+. Ba2+, unlike Ca2+, depolarises the membrane potential by approximately 40 mV, due to rapid block of an inwardly rectifying K+ current. Although Ba2+ permeates CRAC channels at very negative potentials in patch clamp recordings, Ba2+ permeation is steeply voltage-dependent. This combination of Ba2+-dependent depolarisation and voltage-dependent Ba2+ permeation accounts for the apparent lack of Ba2+ permeation through store-operated channels seen in fluorescence experiments. Our findings identify major limitations with the use of Ba2+ as a surrogate for Ca2+ in probing Ca2+ entry pathways and raise the possibility that some of the previous reports proposing multiple Ca2+ entry pathways based on Ba2+ entry into fura 2-loaded cells could be explained by voltage-dependent Ba2+ permeation through CRAC channels.
Bakowski
Daniel
D
Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
Parekh
Anant B
AB
D000818:Animals; D001464:Barium / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism
D015220:Calcium Channels; D005456:Fluorescent Dyes; D015221:Potassium Channels; D001464:Barium; D002118:Calcium; D016257:Fura-2