Essential role of the LIM domain in the formation of the PKCε-ENH-N-type Ca2+ channel complex

A LIM domain is a specialized double-zinc finger motif found in a variety of proteins. LIM domains are thought to function as molecular modules, mediating specific protein-protein interactions in cellular signaling. In a recent study, we have demonstrated that ENH, which has three consecutive LIM domains, acts as an adaptor protein for the formation of a functional PKC epsilon-ENH-N-type Ca2+ channel complex in neurons. Formation of this complex selectively recruits PKC epsilon to its specific substrate, N-type Ca2+ channels, and is critical for rapid and efficient potentiation of the Ca2+ channel activity by PKC in neurons. However, it is not clear whether changes in the local Ca2+ concentrations near the channel mouth may affect the fori-nation of the triprotein complex. Furthermore, the molecular determinants for the interactions among these three proteins remain unknown. Biochemical studies were performed to address these questions. Within the physiological Ca2+ concentration range (0-300 mu M), binding of ENH to the channel C-terminus was significantly increased by Ca2+, whereas increased Ca2+ levels led to dissociation of PKC epsilon from ENH. Mutagenesis studies revealed that the second LIM domain in ENH was primarily responsible for Ca2+-dependent binding of ENH to both the Ca2+ channel C-terminus and PKC epsilon. ENH existed as a dimer in vivo. PKCe translocation inhibition peptide, which blocks the translocation of PKC epsilon from the cytosol to the membrane, inhibited the interaction between PKC epsilon and ENH. These results provide a molecular mechanism for how the PKC epsilon-ENH-N-type Ca2+ channel complex is formed and regulated, as well as potential drug targets to selectively disrupt the PKC signaling complex. (c) 2005 Elsevier Inc. All rights reserved.