A Secretory-type Protein, Containing a Pentraxin Domain, Interacts with an A-type K+ Channel

Dmytro Duzhyy,Margaret Harvey,Bernd Sokolowski

doi:10.1074/jbc.m500111200

Dmytro Duzhyy, Margaret Harvey + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m500111200

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Abstract

A-type K(+) channels belonging to the Shal subfamily are found in various receptor and neuronal cells. Although their kinetics and cell surface expression are regulated by auxiliary subunits, little is known about the proteins that may interact with Kv4 during development. A yeast two-hybrid screening of a cDNA library made from the sensory epithelium of embryonic chick cochlea revealed a novel association of Kv4.2 with a protein containing a pentraxin domain (PPTX). Sequence analysis shows that PPTX is a member of the long pentraxin family, is 53% identical to mouse PTX3, and has a signal peptide at the N terminus. Studies with chick cochlear tissues reveal that Kv4.2 coprecipitates PPTX and that both proteins are colocalized to the sensory and ganglion cells. A yeast two-hybrid assay demonstrated that the last 22 amino acids of the PPTX C terminus interact with the N terminus of Kv4.2. Chinese hamster ovary cells transfected with recombinant PPTX reveal secretory products in both non-truncated and truncated forms. Among the secreted variants are several blocked by Brefeldin A, suggesting export via a classical pathway. PPTX is soluble in the presence of sodium carbonate, suggesting localization to the cytosolic side of the plasmalemma. Immunohistochemical studies show that Kv4.2 and PPTX colocalize in the region of the plasmalemma of Chinese hamster ovary cells; however, both are locked in the endoplasmic reticulum of COS-7 cells, suggesting that PPTX does not act as a shuttle protein. Reverse transcription-PCR demonstrates that PPTX mRNA is found in tissues that include brain, eye, heart, and blood vessels.

Highlights

Ion channels define the excitatory characteristics of sensory and neuronal cells
The pAD-library plasmids were isolated from selected clones, amplified in Escherichia coli strain XL1, and retransformed back to yeast cells containing the vector, pBD-GAL4 Cam, or the plasmid pBD-N, that expressed the N-terminal end of Kv4.2 in fusion with a GAL4 binding domain
PCR was used to amplify the longest part of the protein containing a pentraxin domain (PPTX) cDNA region located next to the pAD-vector sequence, because there was a possibility that the N-terminal sequence was incomplete in these isolated clones

Summary

Introduction

Ion channels define the excitatory characteristics of sensory and neuronal cells. The A-type transient Kϩ channel has a role in this regulation by governing the frequency and amplitude of action potentials [1,2,3]. A yeast two-hybrid screening of a cDNA library made from the sensory epithelium of embryonic chick cochlea revealed a novel association of Kv4.2 with a protein containing a pentraxin domain (PPTX). Sequence analysis shows that PPTX is a member of the long pentraxin family, is 53% identical to mouse PTX3, and has a signal peptide at the N terminus.

Results

Conclusion