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J. Biol. Chem., Vol. 280, Issue 15, 15165-15172, April 15, 2005

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

Dmytro Duzhyy, Margaret Harvey, and Bernd Sokolowski

From the University of South Florida Department of Otolaryngology, Head and Neck Surgery, Department of Physiology and Biophysics, Tampa, Florida 33612

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.

Received for publication, January 4, 2005 , and in revised form, February 10, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY672618.

^* This work was supported by Grant DC43095 from the NIDCD, National Institutes of Health (to B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of South Florida, Otology Laboratory MDC83, Dept. of Otolaryngology-Head and Neck Surgery, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-974-5988; Fax: 813-974-1483; E-mail: bsokolow{at}hsc.usf.edu.

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