Regulation of heterologously expressed transient receptor potential-like channels by calcium ions

Abstract

The Drosophila melanogaster gene product TRPL (transient receptor potential-like) is a Ca 2+-permeable cation channel that contributes to the light-induced Ca 2+ entry in Drosophila photoreceptors and bears homology to several recently cloned mammalian channels. Intracellular Ca 2+ has been implicated to stimulate TRPL channels. This constitutes a potentially dangerous mechanism that may lead to Ca 2+ overload. Therefore, we studied whether TRPL channels, like other Ca 2+-permeable channels, are inhibited by intracellular Ca 2+ concentrations in the micromolar range and whether this effect is mediated by calmodulin. In Sf9 cells expressing the TRPL gene along with histamine H1 receptors after infection with baculoviruses containing the corresponding complementary DNA, histamine-induced TRPL currents were inhibited by intracellular Ca 2+ with an ic 50 of 2.3 μM. Moreover, TRPL currents were reversibly attenuated by a preceding hyperpolarization. This attenuation reflected the action of an increased Ca 2+ influx, since it was abolished in the absence of extracellular Ca 2+ and enhanced by raising extracellular Ca 2+ to 20 mM. Finally, the activity of TRPL channels in inside-out patches was reversibly inhibited by raising the Ca 2+ concentration on the cytosolic side of the patches to 10–50 μM. Addition of calmodulin or the calmodulin inhibitor calmidazolium did not modify the inhibition of the TRPL by Ca 2+. We conclude that high intracellular Ca 2+ concentrations inhibit the TRPL gene, but no evidence was found for the requirement of calmodulin. This mechanism makes Ca 2+ influx through the TRPL gene self-limiting. Furthermore, the TRPL may allow one to study the structural requirements for channel regulation by Ca 2+.