80K-H as a new Ca2+ sensor regulating the activity of the epithelial Ca2+ channel transient receptors potential cation channel V5 (TRPV5)

Abstract

The epithelial Ca2+ channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca2+ entry pathway in the process of active Ca2+ reabsorption. Ca2+ influx through TRPV5 is tightly controlled by modulators of Ca2+ homeostasis, including 1,25-dihydroxyvitamin D3 and dietary Ca2+. However, little is known about intracellular proteins that interact with TRPV5 and directly regulate the activation of this channel. By the use of cDNA microarrays, the present study identified 80K-H as the first protein involved in the Ca2+-dependent control of the epithelial Ca2+ channel TRPV5. 80K-H was initially identified as a protein kinase C substrate, but its biological function remains to be established. We demonstrated a specific interaction between 80K-H and TRPV5, co-localization of both proteins in the kidney, and similar transcriptional regulation by 1,25-dihydroxyvitamin D 3 and dietary Ca2+. Furthermore, 80K-H directly bound Ca2+, and inactivation of its two EF-hand structures totally abolished Ca2+ binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EF-hand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EF-hand pair reduced the TRPV5-mediated Ca2+ current and increased the TRPV5 sensitivity to intracellular Ca2+, accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca2+ sensor controlling TRPV5 channel activity.