External Ca²⁺ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells

Highlights

• The renal epithelial cell line LLC-PK1 expresses endogenous plasma membrane PC2.

• The endogenous PC2 currents are increased by increasing external calcium.

• Calcium-sensing receptor (CaSR) agonists mimic the effect of high calcium.

• PC2 siRNA silencing abolishes the external calcium response.

• A regulatory mechanism links external calcium and PC2 function via activation of CaSR.

Abstract

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel whose dysfunction is associated with the onset of autosomal dominant polycystic kidney disease (ADPKD). PC2 contributes to Ca²⁺ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to the external Ca²⁺ regulation of PC2 channel function. In this study, we explored the effect of external Ca²⁺ on endogenous PC2 in wild type LLC-PK1 renal epithelial cells. We obtained whole cell currents at different external Ca²⁺ concentrations, and observed that the basal whole cell conductance in normal Ca²⁺(1.2 mM), decreased by 30.2% in zero (nominal) Ca²⁺ and conversely, increased by 38% in high external Ca²⁺(6.2 mM). The high Ca²⁺-increased whole cell currents were completely inhibited by either PC2 gene silencing, or intracellular dialysis with active, but not denatured by boiling, PC2 antibody. Exposure of cells to high Ca²⁺ was also associated with relocation of PC2 to the plasma membrane. To explore whether a Ca²⁺ sensing receptor (CaSR) was implicated in the external Ca²⁺ modulation of PC2 currents, we tested the effect of the CaSR agonists, spermine and the calcimimetic R-568, which largely mimicked the effect of high Ca²⁺ under Ca²⁺-free conditions. The CaSR agonist gentamicin also increased the PC2 currents in the presence of normal Ca²⁺. The presence of CaSR was confirmed by immunocytochemistry, which partially colocalized with the intracellular PC2 protein, in an external Ca²⁺-dependent manner. The data support a novel Ca²⁺ sensing mechanism for PC2 expression and functional regulation in renal epithelial cells.

Introduction

PC2 is a member of the superfamily of TRP cation channels [23] that is encoded by the PKD2 gene, whose mutations are responsible for ADPKD [21]. PC2 is a Ca²⁺-permeable nonselective cation channel [10], [36] that is implicated in a number of cellular functions [9]. PC2 has been detected in different cellular locations [7], including the plasma membrane [18], [25], the endoplasmic reticulum [15], [25], and the primary cilium [2], [14], [29]. Ciliary located PC2 in particular, is reported to be one of the key elements in the mechano-sensory response of renal epithelia to fluid flow [24], whose integrity may prevent cyst formation [32]. Previous studies from our laboratory determined a functional PC2 in the primary cilium of LLC-PK1 cells [29], which is regulated by a ciliary located type-2 vasopressin receptor and the production of local cAMP [30]. This PC2 function may have a relevant role in ciliary Ca²⁺ transport, and the regulation of ciliary length. PC2 activation is also associated with a rise in cell Ca²⁺ [24], which may not be mediated by the ciliary PC2 but instead functional channels located in the plasma membrane. Thus, Ca²⁺ influx through a functional plasma membrane PC2 seems to be essential in normal cell responses. Little is known, however, as to the expression and function of endogenous PC2 to the plasma membrane, and how Ca²⁺-dependent mechanisms may regulate this function. This is particularly evident from expected Ca²⁺ feedback signals entailing PC2-mediated Ca²⁺ transport that should depend on the availability of external Ca²⁺. Recent studies from our laboratory, for example, determined that the isolated protein is not affected by extracellular Ca²⁺, but instead to a feedback mechanism mediated by Ca²⁺ influx through the channel, and the regulatory binding of Ca²⁺ to putative intracellular cytoskeletal partners that are controlled by a local pool of Ca²⁺ [3]. PC2 regulatory mechanisms that may be associated with external Ca²⁺ concentrations still are heretofore largely unknown. The fact that PC2 itself does not respond to varying external Ca²⁺ concentrations [3], invokes the need for other sensing mechanisms of external Ca²⁺ regulation that may implicate putative “Ca²⁺ detectors” of extracellular Ca²⁺. One such mechanism is the Ca²⁺-sensing receptor (CaSR) [12], [20] that responds to extracellular Ca²⁺ concentrations in the range of 0.5–10 mM, and has been observed in the various nephron sections of the mammalian kidney [12], [31].

The aim of the present study was to assess whether changes in external Ca²⁺ concentration modify the whole cell conductance of wild type LLC-PK1 renal epithelial cells, and whether this contribution may be associated with endogenous PC2 function. Our data indicate that high external Ca²⁺ increased the whole cell conductance of LLC-PK1 cells particularly the stimulation of endogenous PC2-mediated currents, which were blocked either by silencing of the PKD2 gene, or intracellular dialysis with an active anti-PC2 antibody, raised against the carboxy-terminus of the channel protein. The stimulatory effect of high external Ca²⁺ on the PC2 currents, was mimicked by CaSR agonists, including spermine, and the calcimimetic R-568, in the absence of external Ca²⁺, and gentamicin in the presence of normal Ca²⁺. CaSR was identified by immunocytochemistry, and observed immuno-colocalized with the PC2 channel protein in a manner that depended on external Ca²⁺. The encompassed data are consistent with a regulatory pathway where external Ca²⁺ modulates a Ca²⁺ sensing mechanism that effects PC2 regulation in LLC-PK1 renal epithelial cells. This regulatory mechanism may help explain the connection between Ca²⁺ signals and the onset of ADPKD.