Ion channels of the cyclic nucleotide-binding domain (CNBD) family play a crucial role in the regulation of key biological processes, such as photoreception and pacemaking activity in the heart. These channels exhibit high sequence and structural similarity but differ greatly in their functional responses to membrane potential. The CNBD family includes hyperpolarization-activated ion channels (HCN) and depolarization-activated ether-à-go-go (EAG) channels. Structural and functional studies show that the differences in the coupling interface between these two subfamilies' voltage-sensing domain (VSD) and pore domain may underlie their differential response to membrane polarity. However, other structural components may also contribute to defining the polarity differences in activation. Here we focus on the role of the C-terminal domain, which interacts with elements in both the pore and voltage-sensing domains. By generating a series of chimeras involving the C-terminal domain derived from distant members of the CNBD family, we find that the nature of the C-termini profoundly influences the gating polarity of these ion channels. Scanning mutagenesis of the C-linker region, a helix-turn-helix motif connecting the pore helix to CNBD, reveals residues at the intersubunit interface between the C-linkers are crucial for hyperpolarization-dependent activation. These findings highlight the unique and unexpected role of the intersubunit interface of the C-linker region in regulating the gating polarity of voltage-gated ion channels.
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