Mechanism of an inherited form of incomplete achromatopsia

Dimitri Tränkner*, Herbert Jägle# , Reinhard Seifert*, Lindsay T. Sharpe#, U.Benjamin Kaupp*

* FZ-Jülich, Institut für Biologische Informationsverarbeitung, Germany and
# Universitäts-Augenklinik Tübingen

Introduction: In two siblings with autosomal recessively inherited incomplete achromatopsia, two point mutations within the gene of the a-subunit of the cone cGMP-gated channel were identified. The two mutations are located on different alleles and lead to single amino-acid substitutions in the polypeptide. One substitution is located intracellularly between two transmembrane segments and the other one is in the pore region. The cones of the affected siblings are functional, but display decreased contrast sensitivities, elevated detection thresholds and perturbed light adaptation. After in vitro analysis of the mutant channels we are able to correlate the channel properties with the phenotypic presentation of the achromats.
Methods: Plasmids coding either for the wt and mutant a-subunits or for the cone b-subunit were heterologously expressed in HEK293 cells. Ligand sensitivity of the channels was determined with the patch-clamp technique in inside-out patches of HEK293 cell membranes. Macroscopic currents were recorded under symmetric ionic conditions (in mM): 100 KCl, 10 HEPES, 10 EGTA, 24.5 KOH at pH 7.4. Single-channel currents were determined by fitting the sum of two or three Gaussian functions to the all-points histogram of single-channel recordings. The blockage of fully activated channels by extracellular Ca2+ was studied in outside-out patches. Pipette and bath solution contained (in mM): 105 KCl, 45 KOH, 10 HEPES, 10 NTA at pH 7.4 (HCl). The free Ca2+ concentration in the bath was adjusted by adding CaCl2 and determined potentiometrically.
Results: Heterologous expression of the mutant a-subunits in HEK293 cells revealed that only the pore mutant forms functional channels, with altered properties of ion permeation and activation. The mutant conducts larger currents of monovalent ions that are less efficiently blocked by permeating Ca2+. At physiological membrane voltages, the maximal open probability is substantially reduced, and the EC50 value for the activation by cGMP is markedly shifted to higher cGMP concentrations.
Discussion: We speculate, that the mutant cone channels conduct a smaller fractional Ca2+ current. Consequently, at normal dark current amplitudes, less Ca2+ will enter the mutant cones. Low intracellular Ca2+ might lead to a quasi light-adapted state of the photoreceptors, characterized by a reduced light sensitivity. In addition, incorporation of the mutant in synaptic cGMP-gated channels of cones is expected to perturb synaptic transmission.

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