Nonspecific Cation Current Associated with Native Polycystin-2 in HEK-293 Cells

Abstract

Mutations in either PKD1 or PKD2 gene are associated with autosomal dominant polycystic kidney disease, the most common inherited kidney disorder. Polycystin-2 (PC2), the PKD2 gene product, and the related protein polycystin-L, function as Ca 2؉ -permeable, nonselective cation channels in different expression systems. This work describes a nonspecific cation current (I CC ) that is present in native HEK-293 cells and highly associated with a PC2-channel activity. The current is voltage dependent, activating for potentials that are positive to ؊50 mV and inactivating in a few milliseconds. It is sensitive to Cd 2؉ , Gd 3؉ , La 3؉ , SKF96365, and amiloride. After silencing of PC2 by RNA interfering, cells show a reduced current that is restored by transfection with normal but not truncated PC2. Consistently, I CC is abolished by perfusion with an anti-PC2 antibody. Furthermore, heterologous expression of the PC1 cytoplasmic tail significantly increases I CC peak amplitude compared with native cells. This is the first characterization of such a current in HEK-293 cells, a widely used expression system for ion channels. These cells, therefore, could be regarded as a suitable and readily accessible tool to study interactions between native PC2/PC1 complex and other membrane proteins, thus contributing to the understanding of autosomal dominant polycystic kidney disease pathogenesis.

Role of calcium in polycystic kidney disease: From signaling to pathology

“…Moreover, PC2 appears to be able to generate a non-specific voltage-dependent cation current in native HEK293 kidney cells. This current is strongly associated with PC2 activity, and is completely abolished by the depletion of PC2 protein [20] . Taken together these findings suggest that PC1 and PC2 may affect calcium influx from different cellular compartments, including cilium boundaries (cilioplasm), plasma membrane and ER.…”

Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells

“…The signal location image is a frame subtraction (frames prior to the 100 mV depolarizing step subtracted from frames during the step). The cell with the red overlay is the one patch clamped; the presence of signals in adjoining cells presumably reflects the presence of gap junctions between HEK cells in culture (Pelucchi et al, 2006;Del Re and Woodward, 2005). While fluorescence was seen for all constructs tested, none of the FP-voltage sensors gave an optical signal large enough to be detected in 16 trials (Fig.…”

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

“…However, recording the TRPP2 current has been difficult, because TRPP2 is predominantly expressed in the ER instead of the plasma membrane (21,25) and, more importantly, the activation mechanism of TRPP2 is still unknown. For this reason, almost all reported channel activity of TRPP2 is recorded at the single-channel level (19)(20)(21)(26)(27)(28)(29)(30)(31)(32)(33)(34), which has technical difficulties and limits the questions that can be addressed. The lack of a reliable system to record TRPP2 channel current easily has markedly delayed the study of this functionally important channel.…”