Disease-associated mutations in CNGB3 produce gain of function alterations in cone cyclic nucleotide-gated channels.
OBJECTIVE
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.
Bright
Scott R
SR
Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164, USA.
Brown
Travis E
TE
Varnum
Michael D
MD
D000818:Animals; D003117:Color Vision Defects / Q000235:genetics*; D006152:Cyclic GMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007473:Ion Channels / Q000235:genetics*; D008268:Macular Degeneration / Q000235:genetics*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D014982:Xenopus laevis
C415904:CNGB3 protein, human; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D012333:RNA, Messenger; D006152:Cyclic GMP