Loss-of-Function in the Cataract-associated Frame-shift Mutation of Cx46 is Caused by Inefficient Targeting to the Plasma Membrane

Abstract

Abstract: : Purpose: Inherited cataracts have been associated with a frame-shift mutation in the human lens gap junction protein connexin46 (Cx46), Cx46fs380. This mutation generates a protein 31 amino acids longer in which the last 87 amino acids are different from those of the wild type protein. This mutant has an impaired ability to induce gap junctional currents, acting as a loss-of function mutation when expressed in Xenopus oocytes. The present studies were performed to characterize the cellular and functional abnormalities caused by the carboxyl tail of this mutant. Methods: A series of constructs including Cx46, Cx46fs380, chicken Cx56, Cx56 truncated after amino acid 445 (Cx56tr445), human Cx46 truncated after amino acid 379 (Cx46tr379), and chimeras containing the first 445 amino acids of Cx56 followed by the carboxyl tail of wild type Cx46 (Cx56-445-wtCx46 tail) or Cx46fs380 (Cx56-445-fs380 tail) were expressed in HeLa or NRK cells. Protein expression and distribution were studied by immunoblotting and immunofluorescence. Currents of hemi-gap junction and gap junction channels were recorded by the two-microelectrode voltage-clamp technique in cRNA-injected single or paired Xenopus oocytes. Results: Cx56tr445 and Cx56-445-wtCx46 tail induced both hemichannel and gap junctional currents in oocytes. The Cx56-445-fs380 tail chimera also induced hemichannel and gap junctional currents. However, the amplitude of these currents was significantly reduced. No significant changes in voltage-dependent gating were observed among the different constructs. In the transfected cells, a substantial proportion of the immunoreactivity corresponding to Cx46, Cx56, Cx56tr445, Cx46tr379 or Cx56-445-wtCx46 tail was observed at appositional membranes. In contrast, most of the immunoreactivity to Cx46fs380 and Cx56-445-fs380 tail was observed intracellularly; this intracellular staining showed significant co-localization with the 58K-Golgi protein. Conclusions: The amino acid sequence of the carboxyl tail of Cx46fs380 leads to inefficient targeting of the protein to the plasma membrane and to its co-localization with a Golgi compartment. This abnormal behavior may lead to cataract formation by decreasing intercellular communication.

Keywords: gap junctions/coupling • cell-cell communication • proteins encoded by disease genes