A Novel GCAP1 Mutation Linked to Dominant Cone Dystrophy

Abstract

Purpose:: To elucidate the biochemical characteristics of a novel guanylate cyclase-activating protein 1 (GCAP1) mutation (N104K), which has been identified in a five-generation family associated with autosomal dominant cone dystrophy.

Methods:: GUCA1A gene fragments were amplified from DNA isolated from blood samples of affected and unaffected family members. Amplicons were screened by DHPLC and directly sequenced. WT GCAP1 and GCAP1(N104K) were expressed in insect cells, and assayed for Ca²⁺-dependent GC stimulation, Ca²⁺-dependent mobility shift, and both were subjected to limited proteolysis.

Results:: A novel C312A transversion in exon 2 of the human GUCA1A gene replaced N104 in GCAP1 with K. The mutation N104K is located in the third EF hand motif (EF3) of GCAP1. EF3 was previously shown to be instrumental in converting GCAP1 from a GC inhibitor in the Ca²⁺-bound form to an activator in the Ca²⁺-free form. GCAP1(N104K) displayed only a minor mobility shift at high [Ca²⁺] on SDS PAGE suggesting altered Ca²⁺ binding properties. Limited proteolysis of GCAP1(N104K) showed reduced protection of the mutant at high [Ca²⁺] compared with WT GCAP1 indicating that the mutant GCAP1 can not assume the inhibitory conformation. The mutant GCAP1(N104K) persistently stimulated GC even at high physiological [Ca²⁺], similarly as observed for GCAP1(Y99C), a mutation that also affects EF3.

Conclusions:: The replacement of N104 by K in GCAP1 is the first naturally occurring mutation in the EF3 loop of GCAP1. Our results show that the mutation does not allow stabilization of the Ca²⁺- bound inactive form of GCAP1. The consequence of the mutation is reduced Ca²⁺ binding, persistent stimulation of GC, and cone photoreceptor degeneration.

Keywords: photoreceptors • signal transduction • degenerations/dystrophies