Interaction of Calmodulin with the EAG1 Potassium Channel

The KCNH potassium channel family includes EAG (ether-a-go-go), ERG (eag-related gene) and ELK (eag-like K+) channels. These channels are voltage-gated and are involved in important physiological roles as cardiac repolarization, neuronal excitability, tumor proliferation and hormone secretion. Each channel subunit includes six transmembrane helices and large N- and C-terminal cytoplasmic domains.The cytoplasmic regions include a PAS domain at the N-terminus and a cyclic nucleotide binding-homology domain (CNBhD) at the C-terminus. Recently, it was shown in vivo by FRET that the PAS domain interacts with CNBhD. A structure of this complex contributed further evidence that this interaction is important for channel regulation.Calcium inhibits EAG1 currents; this effect is mediated by calmodulin binding to the channel. Three calmodulin binding sequences have been identified in the human EAG1: BDN, after the PAS domain; BDC1 and BDC2, after the CNBhD. Channels with disrupting mutations at BDN, BDC1 and BDC2 are nearly insensitive to Ca2+/calmodulin, supporting the functional importance of all three sequences for Ca2+/calmodulin regulation. However, a FRET study raised doubts about the physiological relevance of the BDC1 site since channels with mutated BDN and BDC2 no longer interact with calmodulin. In accordance, X-ray structures, both of the CNBhD alone and of the PAS-CNBhD complex reveal that BDC1 is partially occluded.Strikingly, despite everything known about calmodulin, the molecular details on how calmodulin binding results in functional changes in channels and other proteins are for most cases unknown.Here I present biochemical and structural characterization of the interaction of calmodulin with the EAG1 cytoplasmic regions. Using calorimetry and X-ray crystallography I have determined that the three calmodulin BDs have very distinct binding modes to calmodulin and that the presence of the globular domains PAS and CNBhD affects the properties of the calmodulin binding sequences.