DESIGN OF CHIMERIC ION CHANNELS TO MONITOR CAMP-INDUCED CONFORMATIONAL CHANGES AND DYNAMICS

Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are the molecular correlate of Ih (or If) current, which plays a key role in the control of neuronal and cardiac rhythmicity. HCNs are activated by the hyperpolarization of the membrane potential and further regulated by the direct binding of cyclic AMP (cAMP) to the cytoplasmic Cyclic Nucleotide Binding Domain (CNBD). cAMP binding determines the removal of the autoinhibitory action exerted by the CNBD on the pore opening and causes conformational changes that propagate from the CNBD to the pore through the C-linker, increasing channel open probability and speeding activation kinetics. cAMP modulation of HCN controls rhythmicity and excitability at cardiac and neuronal level and pain perception. Thus, in order to understand several physiological functions, as well as diseases affecting both the cardiac and neuronal system, a detailed description of the cAMP-induced conformational changes is required. Moreover, functional studies indicate that cAMP binding to the CNBD induces the transition of the C-terminal domains, from dimer of dimer to tetramer and that this transition is driven by the movements of the C-linker. The goal of this work is to describe the propagation of the movements through the C-linker to the pore. To this end, I have applied spectroscopic techniques, such as Electron Paramagnetic Resonance (EPR) and Double Electron-Electron Resonance (DEER) to study dynamic changes in the structure of the purified and labelled protein. First, I constructed a chimeric channel by fusing the C-linker/CNBD of human HCN4 to the prokaryotic pore domain of KcsA. This protein has the advantage that can be produced and easily purified in E. coli. Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC) measurements demonstrated that the purified chimera is able to bind cAMP. Particularly, ITC gave a Kd value of 1.7 μM, which agrees with the one previously published for the isolated C-linker/CNBD fragment of HCN4. Moreover, the rescue of a K+-uptake deficient E. coli strain demonstrated that the chimeric channel is able to conduct a K+ ions flow. EPR-DEER experiments performed on the chimera revealed that the binding of cAMP to the CNBD domain causes clear conformational changes in the C-terminal region, which transits from a dimer of dimers conformation to a 4-fold symmetrical gating ring. These data confirm previous biochemical and functional indication obtained on the full-length channel and give further details on the direction and the extent of subunit displacement occurring during the conformational transition. The transition of the channel from a dimer of dimer to a tetrameric configuration of the cytosolic domain might reflect a pre-conditioning state for cAMP action on channel gating and partly explains the agonist activity exerted by cAMP on channel kinetics and open probability.