Investigations on structure and functional properties of CNG channels

Ion channels are allosteric proteins that play a fundamental role for cells; they regulate ion fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open) or inactivated (closed). In addition to selecting permeant ions, channels undergo conformational changes that open and close their ion-permeable pores, a process referred to as gating. On the basis of their primary structure the ion channels are classified into a relatively small number of discrete gene families. Cyclic nucleotide gated (CNG) and hyperpolarization-activated, cyclic nucleotide-gated (HCN), together with K+, Na+, Ca2+ and transient receptor potential (TRP) channels belong to the superfamily of voltage gated channels. All members of this family are tetrameric or pseudotetrameric and share a common pore domain that contains two transmembrane (TM) segments - referred to as M1 and M2, or S5 and S6. As in all the voltage-gated channels, CNG channels have four additional TM segments per subunit: S1–S4 that, correspond to the sensor for the allosteric modulation of gating by membrane voltage. CNG channels are mainly activated by binding of cyclic nucleotides, but, in the presence of symmetrical ionic conditions, current-voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion and has been suggested that both the pore and S4 helix contribute to the observed rectification. CNG channels are essential for visual and olfactory transduction: in rod photoreceptors CNG channels open in dark conditions and are responsible for a steady inward current that flows through them keeping the membrane depolarized. In vertebrates, seven members of the CNG channel gene family have been identified that, according to sequence similarity, are grouped into two subtypes, CNGA (CNGA1-CNGA5) and CNGB (CNGB1 and CNGB3). Only CNGA subunits - except the A4 subunit - can form cyclic nucleotide-activated homomeric channels, while CNGB1 and CNGB3 are modulatory and cannot form functional homomeric channels. CNG channels are only slightly voltage-dependent and, for opening, require the binding of cGMP or cAMP to a receptor site located in their C-terminal. In the presence of a saturating agonist, CNG channel gating is voltage independent and it is not known why CNG channels are voltage-insensitive despite harbouring the S4-type voltage sensor. Moreover, unlike many K+ channels, cyclic nucleotide-gated (CNG) channels are known not to inactivate. While electrophysiology combined with mutagenesis have identified the channel pore and the binding domain for cyclic nucleotides (CNs), conformational changes associated with gating have remained elusive. During my PhD course I focused my energies on investigating the structural and functional properties of different domains of the CNGA1 channels, combining results from different techniques like homology modelling, single molecule force spectroscopy (SMFS), molecular dynamics (MD) simulations, and X-ray crystallography with my own data from electrophysiology and site-directed mutagenesis in order to get better insights of fundamental properties of the channels, like permeation and channel gating. The main findings of my PhD thesis are the following: I. I showed that current rectification, in presence of large organic cations, like ethylammonium and dimethylammonium, strongly depends on two voltage-dependent transitions, of which, only the first, is sensible to mutation of charge residues in the S4 helix. These results indicate the existence of at least two distinct mechanisms underlying rectification in CNG channels: a restricted motion of the S4 helix, together with an inefficient coupling to the channel gate that render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na+ and K+. II. has been demonstrated that, contrary to previous belief, when extracellular pH is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. This mechanism is reminiscent of the C-type inactivation observed in K+ channels. Low pH inactivation may represent an unrecognized endogenous mechanism that regulate CNG physiological functions in diverse tissues. III. combining electrophysiology with molecular dynamics (MD) simulations, and X-ray crystallography has been demonstrated that the pore of CNG channels is highly flexible and, mainly due to the side chain of Glu66 in the selectivity filter and to the prolines in the outer vestibule, capable to coordinate different ionic species. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels. IV. by mean of electrophysiology combined with single-molecule force spectroscopy (SMFS), mutagenesis and bioinformatics have been obtained better insights about conformational changes associated with gating and, in particular, about the mechanical coupling between the voltage sensor and the binding domain. We revealed putative electrostatic interactions between an aspartate in the C-linker and an arginine in the S4-S5 linker. We hypothesized a possible mechanism, possibly shared with HCN channels, by which these electrostatic interactions determine the motion of S5, leading to conformational rearrangements of residues flanking the pore so that the lumen of the pore widens and the channel opens.