Sperm GIRK2-containing K+ inward rectifying channels participate in sperm capacitation and fertilization.
ABSTRACT The GIRK2-containing inward-rectifying K(+) ion channels have been implicated in mammalian spermatogenesis. While the Girk2 null mice are fertile, the male weaver transgenic mice carrying a gain-of-function mutation in the Girk2 gene are infertile. To establish the exact period of spermatogenesis affected by this mutation, we performed StaPut isolation and morphological characterization of the germ cells present in the weaver testis. Germ cells representing all periods of spermatogenesis were identified. However, no spermatozoa were present, suggesting that this mutation only affected the haploid phase of spermatogenesis. Real-time PCR studies performed on StaPut purified germ cells from wild-type mice indicated that the Girk2 transcripts were exclusively expressed in spermatids. Immunofluorescence studies of mouse and boar spermatids/spermatozoa localized the GIRK2 K(+) containing channels to the acrosomal region of the sperm plasma membrane. During porcine in vitro fertilization (IVF), GIRK2-containing channels remained associated with the acrosomal shroud following zona-induced acrosome reaction. Fertilization was blocked by tertiapin-Q (TQ), a specific inhibitor of GIRK channels, and by anti-GIRK2 antibodies. Altogether, studies in two different mammalian species point to a conserved mechanism by which the GIRK2 inward-rectifying K(+) ion channels support sperm function during fertilization.
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ABSTRACT: G-protein activated inwardly rectifying K(+) channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate I(K+ACh) in atrium and are regulated by cAMP dependent protein kinase (PKA). Phosphorylation of GIRK1/GIRK4 complexes promotes the activation of the channel by the G-protein Gβγ-dimer ("heterologous facilitation"). Previously we reported that 3 serines/threonines (S/Ts) within the GIRK1 subunit are phosphorylated by the catalytic subunit of PKA (PKA-cs) in-vitro and are responsible for the acute functional effects exerted by PKA on the homooligomeric GIRK1(F137S) (GIRK1(⁎)) channel. Here we report that homooligomeric GIRK4(WT) and GIRK4(S143T) (GIRK4(⁎)) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1(S385CS401CT407C)/GIRK4(WT) composition, where the GIRK1 subunit is devoid of PKA mediated phosphorylation, exhibited reduced but still significant acute effects (reduction during agonist application was≈49% compared to GIRK1(WT)/GIRK4(WT)). Site directed mutagenesis of truncated cytosolic regions of GIRK4 revealed four serines/threonines (S/Ts) that were heavily phosphorylated by PKA-cs in vitro. Two of them were found to be responsible for the acute effects exerted by PKA in vivo, since the effect of cAMP injection was reduced by≈99% in homooligomeric GIRK4(⁎T199CS412C) channels. Coexpression of GIRK1(WT)/GIRK4(T199CS412C) reduced the acute effect by≈65%. Only channels of the GIRK1(S385CS401CT407C) / GIRK4(T199CS412C) composition were practically devoid of PKA mediated effects (reduction by≈97%), indicating that both subunits contribute to the heterologous facilitation of I(K+ACh).Biochimica et Biophysica Acta 01/2013; 1828(4). DOI:10.1016/j.bbamem.2012.12.016 · 4.66 Impact Factor