A. Inanobe et al., Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis, J PHYSL LON, 521(1), 1999, pp. 19-30
1. One of the features of weaver mutant mice is male infertility, which sug
gests 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 an
d electrophysiological techniques.
2. Testicular membrane contained a protein that was recognized by the antib
ody specific to the C-terminus of Kir3.2c (aG2C-3). Its molecular mass was
similar to 45 kDa, which was smaller than that of Kir3.2c (similar to 48 kD
a). The immunoprecipitant obtained from testis with aG2C-3 contained a sing
le 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 fra
me encoding 407 amino acids, whose molecular mass was calculated to be simi
lar to 45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N-ter
minal end, which was the only difference between the two clones. The 18 ami
no acid region possesses the epitope for aG2N-2.
4. In heterologous expression systems of both Xenoypus oocytes and mammalia
n 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 exclusivel
y in the acrosomal vesicles of spermatids, while Kir3.1. immunoreactivity w
as diffuse in the spermatogonia and spermatocytes. These results indicate t
he 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.