PgmNr C53: Polycystin-2 (Pkd2) and its unexpected role in Mg2+ permeability in Paramecium.

Authors:
M. S. Valentine; J. Yano; J. L. Van Houten


Institutes
University of Vermont, Burlington, VT.


Abstract:

The non-selective cation channel polycystin-2 (Pkd2) is a member of the transient receptor potential (TRP) family of proteins and when mutated, can lead to autosomal dominant polycystic kidney disease.  Here, we use the ciliated cell Paramecium tetraurelia, to examine Pkd2 in both the cilia and the cell membrane, together or separately, using deciliated or intact cells.  Paramecium has two paralogs for Pkd2: PKD2a and PKD2b, which are 85% identical at the nucleotide level. Depletion of the mRNA for these paralogs using RNA interference (RNAi) results in reduced backward swimming, specifically in Mg2+ solutions, and a resistance to heavy metal toxicity. Our results suggested Pkd2 may play a role in the Ca2+-dependent Mg2+ pathway because the results are similar to the phenotype of eccentric (XntA1), a mutant that is defective in Mg2+ conductance.   We localized the Pkd2 and XntA proteins by expressing the genes using a plasmid containing a 3’ terminal 3×FLAG or 3×myc sequence.  The Pkd2-FLAG and XntA-FLAG (or XntA-myc) proteins localize in the cell membrane and in the cilia, as confirmed by immunoprecipitation (IP), immunofluorescence, and Western blot.  Using FLAG and myc affinity agarose, we IP’d the epitope-tagged versions of the proteins and demonstrated that XntA-myc co-IPs Pkd2-FLAG, but not vice versa.  These data suggest a potential C-terminal interaction of the Pkd2-FLAG protein with XntA-myc, where the FLAG epitope is probably masked by the Pkd2 protein.  To tease apart the contributions of the Pkd2 channel at the cell membrane and in the cilia, we utilized electrophysiology and membrane potential measurements to assess the permeability of Paramecium’s membrane to Mg2+.  Our data suggest that Pkd2 is both necessary and sufficient for Mg2+ permeability of the cell membrane and that Pkd2 in the cilia may require the XntA protein; that without XntA, Pkd2 cannot function in the cilia.  We propose that the Pkd2 channel is responsible for membrane permeability to Mg2+ while the role of the XntA protein, which is still under investigation, appears to be important for channel positioning or communication among channels in the membrane.

This project was supported in part by NIH Grant Numbers GM59988 and 5 P30 RR032135 and NIGMS grant 8 P30 GM103498.  DNA sequencing performed in the VT Cancer Center supported in part by IDeA from NIGMS of NIH under grant P20GM103449.