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Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'- O -(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn 2+ , and 50 µM Cu 2+ . Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC 50 for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca 2+ and 2 mM Mg 2+ , and 33 µM in low-Mg 2+ , nominally Ca 2+ -free saline. Overall, the results are most consistent with activation of a P2X 7 receptor by ATP 4– . However, low permeability to N -methyl- D -glucamine + and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X 7 receptor activation. ATP stimulation of internalization occurs in Na + -free, Ca 2+ -free, or low-Mg 2+ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X 7 receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp Address for reprint requests and other correspondence: A. K. Ritchie, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, 301 Univ. Boulevard, Galveston, TX 77555-0641 (E-mail: aritchie@utmb.edu )
AJP - Cell Physiology – The American Physiological Society
Published: Oct 1, 2004
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