80K-H as a new Ca2+ sensor regulating the activity of the epithelial Ca2+ channel transient receptor potential cation...

The epithelial Ca(2+) channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca(2+) entry pathway in the process of active Ca(2+) reabsorption. Ca(2+) influx through TRPV5 is tightly controlled by modulators of Ca(2+) homeostasis, including 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). 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 Ca(2+)-dependent control of the epithelial Ca(2+) 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 Ca(2+). Furthermore, 80K-H directly bound Ca(2+), and inactivation of its two EF-hand structures totally abolished Ca(2+) 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 Ca(2+) current and increased the TRPV5 sensitivity to intracellular Ca(2+), 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 Ca(2+) sensor controlling TRPV5 channel activity.