Abstract
As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.
Highlights
As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is called TRPP3 and has similar membrane topology as voltage-gated cation channels
Through the use of mutagenesis, non-reducing SDS-PAGE, blue native PAGE (BN-PAGE) and electrophysiology, among others, we discovered that a novel C-terminal domain called C1 (K575-T622) is essential for both homotrimerization and channel function, challenging the speculation that a functional homotetrameric TRP channel is formed through two consecutive steps of homodimerization
We found that in the kidney, testis and brain under the non-reducing condition, endogenous PKD2L1 displays similar oligomeric patterns from which the band sizes suggest the presence of either PKD2L1 homodimers, -trimers and –tetramers, or its heteromerization with endogenous interacting partner proteins (Fig. 1A)
Summary
A novel PKD2L1 C-terminal domain critical for trimerization and channel function. Wang Zheng1*, Shaimaa Hussein1*, JungWoo Yang[1], Jun Huang[2], Fan Zhang[3], Samuel Hernandez-Anzaldo[4], Carlos Fernandez-Patron[4], Ying Cao[3], Hongbo Zeng[2], Jingfeng Tang5 & Xing-Zhen Chen[1,5]. At least when co-expressed in HEK cells, PKD2L1 interacts with PKD1L3 such that both efficiently traffic to the surface membrane where they act as an extracellular acidinduced off-response cation channel, ie, activation occurred only after low extracellular pH was removed[15,18] They are coexpressed in mouse circumvallate and foliate papillae only PKD2L1 is found in other taste bud areas, including fungiform and palate taste buds[15,18]. This study showed that cells still respond to 25 mM citric acid solution when co-expressed full-length PKD1L3 with PKD2L1 truncation mutant lacking either CC2 (PKD2L1DCC, M1-E653) or both CC2 and the EF-hand domain (PKD2L1DEF-CC, M1-F621), but little response was observed with PKD2L1 truncation mutant lacking the entire Cterminus (PKD2L1DCT, M1-I560), despite robust cell surface expression This suggests the importance of a domain within I560F621 for channel function[25]. Through the use of mutagenesis, non-reducing SDS-PAGE, blue native PAGE (BN-PAGE) and electrophysiology, among others, we discovered that a novel C-terminal domain called C1 (K575-T622) is essential for both homotrimerization and channel function, challenging the speculation that a functional homotetrameric TRP channel is formed through two consecutive steps of homodimerization
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