EXPRESSION OF TRPC3 IN CHINESE-HAMSTER OVARY CELLS RESULTS IN CALCIUM-ACTIVATED CATION CURRENTS NOT RELATED TO STORE DEPLETION

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly e nhanced Ca2+ influx in response to stimulation of membrane receptors l inked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661-671), we t ested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expre ssion of TRPC3 produced cation currents with little Selectivity for Ca 2+ over Na+. These currents were constitutively active, not enhanced b y depletion of calcium stores with inositol-1,4,5-trisphosphate or tha psigargin, and attenuated by strong intracellular Ca2+ buffering. Iono mycin led to profound increases of currents, but this effect was stric tly dependent on the presence of extracellular Ca2+. Likewise, infusio n of Ca2+ into cell through the patch pipette increased TRPC3 currents . Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studie s on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of i onomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 mu M), however, failed to stimulate chann el activity, even in the presence of calmodulin (0.2 mu M). We conclud e that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-ind uced Ca2+-entry but should not be considered store operated.