Native Polycystin 2 Functions as a Plasma Membrane Ca²⁺-Permeable Cation Channel in Renal Epithelia

Abstract

Mutations in polycystin 2 (PC2), a Ca2؉ -permeable cation channel, cause autosomal dominant polycystic kidney disease. Whether PC2 functions in the endoplasmic reticulum (ER) or in the plasma membrane has been controversial. Here we generated and characterized a polyclonal antibody against PC2, determined the subcellular localization of both endogenous and transfected PC2 by immunohistochemistry and biotinylation of cell surface proteins, and assessed PC2 channel properties with electrophysiology. Endogenous PC2 was found in the plasma membrane and the primary cilium of mouse inner medullar collecting duct (IMCD) cells and Madin-Darby canine kidney (MDCK) cells, whereas heterologously expressed PC2 showed a predominant ER localization. Patch-clamping of IMCD cells expressing endogenous or heterologous PC2 confirmed the presence of the channel on the plasma membrane. Treatment with chaperone-like factors facilitated the translocation of the PC2 channel to the plasma membrane from intracellular pools. The unitary conductances, channel kinetics, and other characteristics of both endogenously and heterologously expressed PC2 were similar to those described in our previous study in Xenopus laevis oocytes. These results show that PC2 functions as a plasma membrane channel in renal epithelia and suggest that PC2 contributes to Ca 2؉ entry and transport of other cations in defined nephron segments in vivo.Polycystins represent an expanding family of membrane proteins composed of two subfamilies, polycystin 1-like and polycystin 2-like molecules. PC1-like molecules consist of polycystin 1 (PC1) (1, 16), polycystin-REJ (15), polycystin-1L1 (40), and polycystin-1L2 (unpublished data), which likely function as unorthodox G protein-coupled receptors (5, 24). PC2-like molecules are ion channels and encompass PC2 (20), polycystin-L (4, 23, 38), and polycystin-2L2 (11). While the disease associations of the other polycystins are unknown, mutations in PC1 and PC2 cause autosomal dominant polycystic kidney disease, the leading genetic cause of renal failure. Formation of a large number of fluid-filled cysts in the kidney is the main characteristic of the disease.PC1, encoded by PKD1, is predicted to be a Ϸ460-kDa integral membrane glycoprotein with a very large extracellular amino terminus, 11 transmembrane domains, and a small intracellular carboxyl terminus (1, 16). PC1 is found in the plasma membrane or in the cell-cell junction of cultured cells and in tissues (8,17,27). PC1 has recently been shown to function as a G protein-coupled receptor (5), although its ligand(s) has not been identified. PC2, the product of PKD2, is predicted to encode an integral membrane protein of Ϸ110 kDa with an EF-hand domain at its carboxyl terminus (20). Sequence homology to other ion channels suggests a pore-forming capacity of PC2. Since mutations in PC1 and PC2 result in similar phenotypes and PC2 is able to interact with PC1 through its coiled-coil domain in vitro (28,34), it has been speculated that PC1 and PC2 form a functional complex. Ho...

Cell biology of polycystin-2

“…While this question has not been formally addressed, there are several lines of evidence to suggest the former. In this regard, biochemical experiments have shown that only a small fraction of transfected PKD2 (>5%) reached the plasma membrane [36], in agreement with a previous report that native or transfected PKD2 could be barely detected in the plasma membrane of LLC-PK1 cells [24]. Therefore, the modest increase in whole cell currents in the Ma et al study [36] could simply reflect low presence of PKD2 in the plasma membrane.…”

“…In terms of their subcellular localizations, TRP channels have been localized in various subcellular sites such as plasma membrane [12], endoplasmic reticulum [16], lysosomes [17][18][19], and primary cilium [20][21][22]. Consistently, PKD2 expression has been reported in the plasma membrane [23,24], ER [25], primary cilium [26], centrosome [27], and mitotic spindles in dividing cells [28]. Specific functions of PKD2 have been associated with its expression in the plasma membrane, ER, and primary cilium (Fig.…”

“…It is noteworthy that bending-induced Ca 2+ signaling was not affected by inhibition of ryonodine receptors [55]. Several groups have shown the expression of PKD1 and PKD2 in the primary cilium [24,26,58,59]. Recently Geng et al [59] identified an N-terminal R 6 VxP motif responsible for PKD2 targeting to the cilium.…”

“…It allowed non-selective passing of cations, with slightly higher selectivity for Ca 2+ over Na + and K + , but higher conductance in K + [34]. However, the high single channel conductance (100-200 pS) [24,31,35] in combination with the relatively high open probability (~20-40%) [24,31,35] did not correlate well with the moderate amplitude of whole cell currents (several hundreds pA at -100 mV) in cells overexpressing PKD2 [35,36]. This, along with the observation that the vast amount of PKD2 was in the ER [25] raised the question of whether PKD2 alone or even in combination with PKD1 could actually form a functional channel in the plasma membrane.…”

TRP channels as potential drug targets

“…PKD2 is a non selective cation channel and loss of function has been implied to cause polycystic kidney disease (Luo et al, 2003). The activity of the channel is influenced by the gene product of PKD1 which directly interacts with the C-terminal of PKD2 (Tsiokas et al, 1997;Xu et al, 2003, Vandorpe et al, 2002.…”