Connexin structure, interaction with kinesin and Schwann cell communication

Abstract

X-linked Charcot-Marie-Tooth (CMTX) disease is characterized by peripheral nerve demyelination, due to expression of aberrant forms of the gap junction protein connexin32 (Cx32) in Schwann cells. Loss of Cx32 function causes demyelination, even in the continued presence of intercellular communication resulting from expression of other connexins. Our central hypothesis is that demyelination is due to the loss of connexin-protein interactions, involving protein misfolding, mislocalization or altering expression of interacting proteins.;We have explored the underlying structural propensity of three connexin proteins by quantifying helical secondary structure content using circular dichroism spectroscopy of synthetic peptides corresponding to cytoplasmic domains of Cx32, Cx36 and Cx43, in aqueous buffers and 30% trifluoroethanol (TFE). We conclude that several peptides in 30% TFE demonstrate helical features, representing common regions in all three connexins. Some CMTX mutations fall within these structured regions, suggesting that mutation-induced structural alterations could have functional consequences.;We next studied the interaction of Cx32 with kinesin, a cytoskeletal motor. Our hypothesis is that part of connexin delivery to the plasma membrane is mediated by interaction with microtubule-motors. Biochemical characterization of Cx32 from liver tissue showed direct kinesin-Cx32 interaction in a region with helical structure and where some CMTX mutations show decreased Cx32 trafficking. Cx32-vesicle motility experiments confirmed kinesin's role.;In order to determine the effects of Cx32 protein loss, we studied Schwann cells from wild-type and connexin-null mice. We found that Cx32-null Schwann cells still maintained a high degree of communication. However, calcium wave propagation upon mechanical stimulation was markedly reduced. We also found changes in cell response to ATP-analogues and ATP-receptor antagonists, suggesting switch of ATP receptor subtypes. Since ATP is known to play a role in nerve-Schwann cell communication, it is possible that Cx32 is necessary for maintenance of myelin.;We have found similar secondary structural features in three connexins. One structured region binds a trafficking motor. Loss of connexin function may affect expression of other proteins in the nexus complex, thereby affecting cell physiology. In studying these three elements of protein function, folding, trafficking and interactions, we hope to have added to the understanding of connexin function.