Potassium channels involved in human sperm volume regulation--quantitative studies at the protein and mRNA levels

KCNE1, KCNA5 and KCNK5 have been identified, by using specific blockers, as K(+)-channels involved in sperm volume regulation under physiological conditions. All three channels were localised on the cytoplasmic droplets and tail of human ejaculated spermatozoa by fluorescence microscopy. Using flow cytometric quantification, KCNE1 was found to be present in 80% or more spermatozoa and KCNK5 in only about 20%, with KCNA5 expressed by 20-90% of cells. Whereas the extents of such protein expression did not differ statistically between semen donors and subfertile patients, the former group exhibited higher capacities for sperm volume regulation which were correlated with other sperm qualities including normal morphology and motile sperm number in the ejaculate. Channel identification was further confirmed at the protein level using Western blotting. RT-PCR analysis of testicular and sperm RNA of proven quality indicated the presence of Kcne1, Kcna5 and Kcnk5 transcripts. Subsequent sequencing of PCR products demonstrated that the nucleotide sequences of the entire encoding regions of Kcne1 and Kcnk5 were identical to those published in the database, whereas that of Kcna5 mRNA showed a single nucleotide synonymous deviation that agrees with the published genomic sequence. Quantitative real-time PCR analysis of sperm RNA revealed the amounts of Kcne1 > Kcna5 > Kcnk5, in the same order as for protein expression. Thus, KCNE1 is probably the major K(+)-channel involved in regulatory volume decrease in human spermatozoa, and channel activity is regulated beyond the extent of protein expression.