Receptor for Activated C Kinase 1 (RACK1) Inhibits Function of Transient Receptor Potential (TRP)-type Channel Pkd2L1 through Physical Interaction

Pkd2L1 (also called TRPP3) is a non-selective cation channel permeable to Ca2+, Na+, and K+ and is activated by Ca2+. It is also part of an acid-triggered off-response cation channel complex. We previously reported roles of the Pkd2L1 C-terminal fragments in its channel function, but the role of the N terminus remains unclear. Using a yeast two-hybrid screening, we found that the Pkd2L1 N terminus interacts with the receptor for activated C kinase 1 (RACK1), a scaffolding/anchoring protein implicated in various cellular functions. This interaction requires the last two Trp-Asp (WD) motifs of RACK1 and fragment Ala19–Pro45 of Pkd2L1. The interaction was confirmed by GST pulldown, blot overlay, and co-immunoprecipitation assays. By 45Ca tracer uptake and two-microelectrode voltage clamp electrophysiology, we found that in Xenopus oocytes with RACK1 overexpression Pkd2L1 channel activity is abolished or substantially reduced. Combining with oocyte surface biotinylation experiments, we demonstrated that RACK1 inhibits the function of Pkd2L1 channel on the plasma membrane in addition to reducing its total and plasma membrane expression. Overexpressing Pkd2L1 N- or C-terminal fragments as potential blocking peptides for the Pkd2L1-RACK1 interaction, we found that Pkd2L1 N-terminal fragment Met1–Pro45, but not Ile40–Ile97 or C-terminal fragments, abolishes the inhibition of Pkd2L1 channel by overexpressed and oocyte-native RACK1 likely through disrupting the Pkd2L1-RACK1 association. Taken together, our study demonstrated that RACK1 inhibits Pkd2L1 channel function through binding to domain Met1–Pro45 of Pkd2L1. Thus, Pkd2L1 is a novel target channel whose function is regulated by the versatile scaffolding protein RACK1. Pkd2L1 is a calcium- and acid-activated channel likely involved in acid sensing, but how it is regulated remains unclear. Receptor for activated C kinase 1 (RACK1), known to regulate various receptors/channels, interacts with and inhibits the function of Pkd2L1. Pkd2L1 is a novel target channel of RACK1. Pkd2L1-RACK1 interaction may play important physiological roles through regulating channel activation by calcium or acid.