Ca2+-dependent Conformational Changes in a C-terminal Cytosolic Domain of Polycystin-2

Abstract

The PKD1 and PKD2 genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PKD2 gene codes for a 968-amino acid long membrane protein called polycystin-2 that represents a cation channel whose activity can be regulated by Ca 2؉ ions. By CD, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of polycystin-2, polycystin-2-(680 -796) that was proposed to contain a Ca The PKD2 gene is one of the two genes mutated in patients with autosomal-dominant polycystic kidney disease (1). It encodes polycystin-2, a 968-amino acid-long protein with 6 putative membrane-spanning domains. According to structural predictions, both its N and C termini extend into the cytoplasm; furthermore, a pore-forming region has been postulated between the fifth and sixth membrane-spanning domains. By sequence comparison a coiled-coil domain and a Ca 2ϩ -binding EF-hand have been predicted in the C terminus of polycystin-2 (1) and a homology model has been published recently (2). However, so far only indirect evidence for the presence of these motifs has been presented. Electrophysiological measurements by several groups have demonstrated that polycystin-2 is a non-selective cation channel and that its channel activity is regulated by calcium. One report has shown the activation of polycystin-2 by the addition of 1 M Ca 2ϩ to the bath solution and its inhibition by millimolar concentrations of Ca 2ϩ (3). An R742X mutant protein, however, did not respond to the addition of Ca 2ϩ . These initial observations were confirmed by other publications that reported that Ca 2ϩ concentrations of up to 1.26 mM increase the probability for the open state of full-length polycystin-2 (4), whereas higher concentrations are inhibitory (4, 5). Again, the activity of a truncated protein, this time a 703-amino acid mutant, is not modulated by calcium (4).From the above it seems obvious that the C terminus via binding of Ca 2ϩ exerts a modulatory activity on the channel activity of polycystin-2. This part of the protein, however, also is of particular interest because it interacts with a wide variety of other proteins, among them polycystin-1, ion channels (inositol 1,4,5-trisphosphate receptor, polycystin-2 itself, and TRPC1), cytoskeletal proteins (␣-actinin, CD2AP, mDia1, troponin I, and tropomyosin-1), intracellular trafficking proteins (PACS-1, PACS-2, and PIGEA-14) and even a transcription factor (Id2) (6). Whether all of these interactions are direct or require additional cofactors is not clear at present; furthermore, it is not known whether any of them are cooperative or exclusive. We have therefore decided to subject the C terminus of polycystin-2 to an extensive biochemical and structural analysis. 3 The abbreviations used are: DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; CDPK-␣, calmodulin-like domain of the calcium dependent soybean protein kinase ␣; HSQC, heteronuclear single quantum coherence; ISIC, intelligent structural infor...

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

“…Given the rela- tively high [Ca 2ϩ ] predicted at the mouth of the IP 3 R at the ER surface (34,42), a micromolar affinity for Ca 2ϩ may allow TRPP2 to sense such local changes in [Ca 2ϩ ] cyt . Recent studies on the structure of the EF-hand within the cytosolic C terminus of TRPP2 have brought new insights into the conformational changes that occur upon Ca 2ϩ binding (6,8). However, the direct link between Ca 2ϩ binding to the EF-hand of TRPP2 and its Ca 2ϩ -dependent channel regulation has not been demonstrated yet and requires further research.…”

“…Structural analyses indicate that TRPP2 contains several functional domains in its C-terminal tail. There are two Ca 2ϩ -binding sites (aa 680 -796) arranged in a typical and an atypical EF-hand motif, which could be involved in a Ca 2ϩ -mediated regulation of TRPP2 (6). An endoplasmic reticulum (ER) retention signal (aa 787-820) (7) and a coiled-coil domain (aa 839 -919), responsible for homo-and heterodimerization (8,9), are also present.…”

Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca 2+ -dependent regulation of polycystin-2 channel activity

“…R 1 and R 2 NMR Relaxation Rates. T1 and T2 relaxation times were extracted from two series each of 1 H-15 N HSQC spectra with delays of 100, 300, 500, 700, and 1,000 ms for T1 and 10,30,70,150,190, and 250 ms for T2, with a 1-s recycle delay. NMR peak heights determined by the "rh" command in SPARKY, and the program CurveFit (38) was used for exponential fitting of R 1 and R 2 .…”

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant

“…5B). L736 and N737 are located in the C-terminal EF-hand motif, a domain involved in Ca 2+ regulation of TRPP2 activity (77)(78)(79)(80). Although they are not directly involved in Ca 2+ binding (78), deletion of L736 and N737 most likely alters Ca 2+ regulation by deforming the Ca 2+ binding site.…”