Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis.

Abstract

1. One of the features of weaver mutant mice is male infertility, which suggests that Kir3.2, a G-protein-gated inwardly rectifying K+ channel subunit, may be involved in spermatogenesis. Therefore, we have characterized the Kir3.2 isoform in mouse testis using immunological, molecular biological and electrophysiological techniques. 2. Testicular membrane contained a protein that was recognized by the antibody specific to the C-terminus of Kir3.2c (aG2C-3). Its molecular mass was approximately 45 kDa, which was smaller than that of Kir3.2c ( approximately 48 kDa). The immunoprecipitant obtained from testis with aG2C-3 contained a single band of the 45 kDa protein, which could not be detected by the antibody to the N-terminus common to the known Kir3.2 isoforms (aG2N-2). 3. A novel alternative splicing variant of Kir3.2, designated Kir3.2d, was isolated from a mouse testis cDNA library. The cDNA had an open reading frame encoding 407 amino acids, whose molecular mass was calculated to be approximately 45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N-terminal end, which was the only difference between the two clones. The 18 amino acid region possesses the epitope for aG2N-2. 4. In heterologous expression systems of both Xenopus oocytes and mammalian cells (HEK 293T), Kir3.2d either alone or with Kir3.1 exhibited G-protein-gated inwardly rectifying K+ channel activity. 5. Prominent Kir3.2d immunoreactivity in the testis was detected exclusively in the acrosomal vesicles of spermatids, while Kir3.1 immunoreactivity was diffuse in the spermatogonia and spermatocytes. These results indicate the possibility that the testicular variant of Kir3.2, Kir3. 2d, may assemble to form a homomultimeric G-protein-gated K+ channel and be involved in the development of the acrosome during spermiogenesis.