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.

Disease-associated mutations in CNGB3 produce gain of function alterations in cone cyclic nucleotide-gated channels.

PURPOSE: To characterize the functional consequences of disease-associated mutations in the CNGB3 (B3) subunit of human cone photoreceptor cyclic nucleotide-gated channels in order to gain insight into disease mechanisms. METHODS: Three separate disease-associated mutations were generated in CNGB3: F525N, R403Q, and T383fsX. These mutant subunits were then heterologously expressed in Xenopus oocytes in combination with wild type CNGA3 (A3) subunits, and characterized by patch-clamp recording in the inside-out configuration. RESULTS: Co-expression of A3 and B3F525N, A3 and B3R403Q, or A3 and B3R403Q and B3T383fsX subunits resulted in channels that exhibited an increase in ligand sensitivity without a reduction in current density compared to wild-type heteromeric channels. Each simulated disease state produced channels that exhibited greater sensitivity to block by L-cis-diltiazem than homomeric CNGA3 channels, confirming that the mutant CNGB3 subunits were competent to form functional heteromeric channels. Each combination of subunits displayed an increase in apparent affinity for cGMP relative to wild-type heteromeric channels. However, F525N enhanced cGMP apparent affinity to a significantly greater extent than the other two modeled disease states. CONCLUSIONS: We have examined the gating effects of two previously uncharacterized disease-associated mutations in the CNGB3 subunit and found that in each case, the mutations resulted in a gain of function molecular phenotype. Furthermore, the magnitude of the effect on channel function correlated with the severity of the associated disease. The complete achromatopsia-associated F525N mutation resulted in more pronounced alterations in channel function than the mutation combinations linked to macular degeneration or progressive cone dystrophy.