Identification and characterization of a novel splice variant of β3 subunit of GABA A receptor in rat testis and spermatozoa

Abstract

γ-Aminobutyric acid type A (GABA A) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of β3 subunit of GABA A receptor, designated as β3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the β3 subunit of neuronal GABA A receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The β3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the β3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of β3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the β3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the β3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of β3 subunit of GABA A receptor, designated as β3t, is found in germ cells of rat testis and sperm. The inability of the β3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.