Phosphatidylinositol 4,5-bisphosphate activates Slo3 currents and its hydrolysis underlies the epidermal growth factor-induced current inhibition.
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 (PIP(2)). Depletion of endogenous PIP(2) 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 PIP(2)-dependent manner. High concentrations of PIP(2) 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-PIP(2) interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP(2) is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP(2) leads to inhibition of Slo3 currents both in native and heterologous expression systems.
Tang
Qiong-Yao
QY
Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298-0551, USA.
Zhang
Zhe
Z
Xia
Jingsheng
J
Ren
Dejian
D
Logothetis
Diomedes E
DE
D020101:Acrosome Reaction; D000818:Animals; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D008297:Male; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry; D013094:Spermatozoa / Q000378:metabolism*; D014982:Xenopus laevis
C110627:Kcnu1 protein, mouse; D051036:Large-Conductance Calcium-Activated Potassium Channels; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D015221:Potassium Channels; D004815:Epidermal Growth Factor
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