1. Department of Physiology and Biophysics, Howard Hughes Medical Institute, Box 357290, University of Washington School of Medicine, Seattle, Washington 98195-7290, USA
2. Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USA
3. Center for Neurobiology and Behavior, Howard Hughes Medical Institute, Columbia University, 722 West 168th Street, New York, New York 10032, USA
4. Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, New York 10032, USA

Correspondence to: William N. Zagotta¹Eric Gouaux^2,4 Email: jeg52@columbia.edu
Email: zagotta@u.washington.edu
The atomic coordinates for the HCN2I-cAMP, HCN2J-cAMP and HCN2J-cGMP structures described here have been deposited in the Protein Data Bank under the accession codes 1Q43, 1Q50 and 1Q3E, respectively.

Abstract

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration¹. These nonselective cation channels, underlying the I_f, I_h and I_q currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP². The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by -adrenergic agonists³. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.