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The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K+ currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of endogenous PIP2 in inside-out macropatches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor, demonstrated that stimulation by epidermal growth factor (EGF) could inhibit channel activity in a PIP2-dependent manner. High concentrations of PIP2 in the patch pipette not only resulted in a strong increase in sperm Slo3 current density but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP2 interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP2 is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP2 leads to inhibition of Slo3 currents both in native and heterologous expression systems. [ABSTRACT FROM AUTHOR]

Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca2+-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shuck protein 70-2, a testis-specific chaperone, and CatSperβ, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSperβ was restricted to testis and localized to the principal piece of the sperm tail. CatSperβ protein is absent in CatSper1-/- sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSperβ is the first identified auxiliary protein to the CatSper channel. [ABSTRACT FROM AUTHOR]