Ligand-Binding Characteristics of the Cyclic Nucleotide Binding Domain (CNBD) of the HERG Potassium Channel and Long QT Syndrome

Abstract

cAMP may regulate human ether-a-go-go-related gene potassium channel (HERG) by either stimulating protein kinase A (PKA) to phosphorylate the channel or direct binding to the cyclic nucleotide binding domain (CNBD) of HERG. The consequence of direct cAMP binding to HERG is still unclear. Mutations throughout the HERG channel can disrupt its function, leading to the Long QT syndrome. Dysfunction of HERG harboring mutations in the CNBD has been attributed to inability to bind cyclic nucleotides and/or improper trafficking. We characterized ligand binding of HERG potassium channel nucleotide binding domain (CNBD) using surface plasma resonance (SPR), fluorescence and affinity pull down experiments. Recombinant HERG-CNBD was purified and used for these studies. SPR measured cAMP binding to the isolated CNBD domain with estimated Kd value of 14 - 39 μM. Long QT2 mutants (F805S, G816V, S818L V822M and R823W) introduced into the recombinant protein retained binding with cAMP, suggesting that deleterious channel effects of these naturally occurring mutations are not caused by direct disruption of cAMP binding. Progressive deletion/truncation study of HERG-CNBD showed that a 16-amino acid region (residues 755-811) of HERG contributed most to the cAMP binding. Our results confirmed the direct binding of cAMP to HERG CNBD motif and narrow down the region involved. Disruption of ligand binding however does not seem to be a primary mechanism for channelopathy disease. Further studies are needed to determine if CNBD causes conformational change upon cAMP binding leading to channel regulation.