Regulation of human cone cyclic nucleotide-gated channels by endogenous phospholipids and exogenously applied phosphatidylinositol 3,4,5-trisphosphate.
Cyclic nucleotide-gated (CNG) channels are critical components of the vertebrate visual transduction cascade involved in converting light-induced changes in intracellular cGMP concentrations into electrical signals that can be interpreted by the brain as visual information. To characterize regulatory mechanisms capable of altering the apparent ligand affinity of cone channels, we have expressed heteromeric (CNGA3 + CNGB3) human cone CNG channels in Xenopus laevis oocytes and characterized the alterations in channel activity that occur after patch excision using patch-clamp recording in the inside-out configuration. We found that cone channels exhibit spontaneous changes in current at subsaturating cGMP concentrations; these changes are enhanced by application of ATP and seem to reflect alterations in channel gating. Similar to rod CNG channels, lavendustin A prevented this regulation, suggesting the involvement of a tyrosine phosphorylation event. However, the tyrosine residue in CNGB3 (Tyr545) that is equivalent to the critical tyrosine residues in rod and olfactory CNG channel subunits does not participate in cone channel regulation. Furthermore, the changes in ligand sensitivity of CNGA3 + CNGB3 channels were prevented by inhibition of phosphatidylinositol 3-kinase (PI3-kinase) using wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which suggests that phospholipid metabolism can regulate the channels. Direct application of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to the intracellular face of excised patches also resulted in down-regulation of channel activity. Thus, phospholipid metabolism and exogenously applied PIP3 can modulate heterologously expressed cone CNG channels.
Bright
Scott R
SR
Department of Veterinary, Washington State University, PO Box 646520, Pullman, WA 99164, USA.
Rich
Elizabeth D
ED
Varnum
Michael D
MD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D054815:Cyclic Nucleotide-Gated Cation Channels; D004594:Electrophysiology; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D010743:Phospholipids / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D014982:Xenopus laevis
C415904:CNGB3 protein, human; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D011994:Recombinant Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D000255:Adenosine Triphosphate