Identification of a Sequence Responsible For the Abnormal Behavior of a Cataract–Associated Mutant Cx46

Abstract

Abstract: : Purpose: To elucidate the molecular mechanism by which a mutant Cx46, Cx46fs380, leads to cataract formation. Cx46fs380 contains a frame–shift at amino acid 380 resulting in a protein with a carboxyl terminus 31 amino acids longer than wild type Cx46 in which the last 87 amino acids are aberrant. Methods: Chimeric connexins containing different fragments or mutated forms of the aberrant carboxyl terminus or the carboxyl terminus of wild type Cx46 were generated by PCR. The functional and cellular consequences conferred by the aberrant carboxyl termini of the different chimera were assessed in exogenous expression systems. The gap junctional currents induced by the chimeras were measured using double whole–cell voltage–clamp in Xenopus oocyte pairs after RNA microinjection. The cellular distribution of the chimeric proteins was studied by immunofluorescence in transfected cells. Results: Cx46fs380 and chimeras of Cx56 and Cx32 containing the aberrant carboxyl terminus of Cx46fs380 induced very little or no gap junctional currents. These proteins localized intracellularly. RNAs coding for chimeras containing amino acids 1–58 of the aberrant carboxyl terminus induced gap junctional currents in Xenopus oocytes. Cells transfected with these chimeric proteins showed anti–fs380 immunoreactivity at appositional membranes. In contrast, a Cx56 chimera containing the last third of the aberrant carboxyl terminus induced very little gap junctional activity. Moreover, anti–fs380 immunoreactivity showed an intracellular localization in cells transfected with this chimera. Alanine substitution of critical residues in the aberrant carboxyl terminus restored function of Cx46fs380 and the Cx56 and Cx32 chimeras Conclusions: The carboxyl terminus of Cx46fs380 contains a sequence that interferes with trafficking/targeting of the protein to the plasma membrane. In the lens, this would result in decreased intercellular communication and cataract formation.

Keywords: gap junctions/coupling • mutations • cell-cell communication