Ion channels play a fundamental role in the generation of an electrical response to light in rods and cones of the vertebrate retina. The closing of a cation-selective channel in the outer segment of these photoreceptors is the final step in the enzymatic cascade that begins with the absorption of a photon of light by rhodopsin. The photoactivated rhodopsin activates a phosphodiesterase via the GTP-binding protein transducin. The phosphodiesterase catalyzes the hydrolysis of guanosine 3',5'-cyclic monophosphate (cGMP), lowering the cytosolic concentration of cGMP and closing a cGMP-gated channel in the membrane of the outer segment. The closing of a cation-selective channel causes a hyperpolarization of the photoreceptor outer segment that is transmitted to the inner segment, where it modulates transmitter release. Clearly the cyclic nucleotide–gated (CNG) channels play a central role in visual transduction. They play a similarly important role in olfactory transduction and have even been discovered in the hippocampus, where they are postulated to have a role in the synaptic plasticity underlying learning and memory.
CNG channels are part of a larger family of ion channels that are directly regulated by cyclic nucleotides. This family includes the hyperpolarization-activated cyclic nucleotide–modulated (HCN) channels and the EAG-related channels. These channels play a role in a number of other important physiological processes. For example, the HCN channels, sometimes referred to as pacemaker channels, have a role in generating the pacemaker activity of cardiac and neuronal cells. The cyclic nucleotide–dependent regulation of HCN channels is thought to be largely responsible for the increase in heart rate with β-adrenergic agonists such as epinephrine. Therefore, a detailed understanding of the molecular mechanisms of these channels' function provides insight into electrical signaling in a number of sensory and physiological processes.
The cyclic nucleotide–regulated channels are beautifully optimized for their role in cell signaling. Different cyclic nucleotide–regulated channels exhibit different selectivities for cGMP and cAMP, depending on their physiological role. By cooperatively binding multiple cyclic nucleotide molecules, these channels become exquisitely sensitive to changes in the levels of cytosolic cyclic nucleotide, allowing, for example, the rod photoreceptor CNG channel to detect faithfully and signal the drop in cGMP concentration resulting from the absorption of a single photon. Cyclic nucleotide–regulated channels were originally thought to be static sensors of cyclic nucleotide concentration, but it has recently been found that the concentration of nucleotide that opens the channels can be tuned by physiological stimuli such as Ca2+/calmodulin, phosphorylation, phosphoinositides, and transition metal divalent cations. We have focused our research on understanding the molecular mechanisms that underlie these specializations.
Over the past several years, we have used a combination of electrophysiological recording, site-directed mutagenesis, site-specific fluorescence recording, and x-ray crystallography to investigate the molecular mechanisms underlying the cyclic nucleotide–dependent regulation of these channels. While I was on sabbatical in Eric Gouaux's laboratory (HHMI, Columbia University College of Physicians and Surgeons), we determined the structure of a carboxyl-terminal fragment of HCN2, a cyclic nucleotide–regulated channel expressed in the heart and brain. The carboxyl-terminal region contains the cyclic nucleotide–binding domain (CNBD) and C-linker region which connects the CNBD to the pore and exhibits strong sequence similarity in all of the cyclic nucleotide–regulated channels. Our structure revealed a novel tetramerization domain, mechanism of cyclic nucleotide specificity, and inter- and intrasubunit interactions that may be important for ligand-dependent channel gating and modulation in all of the cyclic nucleotide–regulated channels.
More recently we solved the x-ray structure of the carboxyl-terminal region of SpIH, a sea urchin channel related to CNG and HCN channels. Like HCN channels, SpIH channels are activated by hyperpolarizing voltage pulses. However, SpIH channels exhibit a large increase in current in response to cyclic nucleotides, a response that is more similar to that of CNG channels. Unlike both CNG and HCN channels, SpIH is activated fully by cAMP but only partially by cGMP. We exploited our new structure and the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of CNG channels and many other cyclic nucleotide–regulated enzymes. Introduction of a threonine into the β roll of the CNBD specifically increased affinity for cGMP, as predicted from our previous HCN2 structure, but cGMP remained a partial agonist. We found, however, that introduction of an aspartic acid into the C-helix of the CNBD caused the SpIH channels to be activated fully by cGMP and only partially by cAMP, similar to CNGA1 channels. Further x-ray crystallography revealed that the aspartic acid formed a pair of hydrogen bonds with the N1 and N2 hydrogens on the guanine ring of cGMP, as we had previously proposed. These results demonstrated the mechanism for cGMP selectivity in CNG channels and probably cGMP-dependent protein kinase as well. They also suggest that these hydrogen bonds form during the allosteric opening transition of the channel, perhaps by a movement of the C-helix.
To measure directly the rearrangements in the CNBD and C-linker that underlie the cyclic nucleotide–dependent activation of the channels, we needed to develop new methods. First we developed patch-clamp fluorometry (PCF), a method that allows the simultaneous measurement of both fluorescence and current from excised inside-out membrane patches, while changing the voltage or applying intracellular regulators. This method allows us to measure dynamic structural changes in channels with fluorescence, while at the same time measuring functional changes in channels with electrophysiology. We then combined this method with fluorescence measurements of inter- and intramolecular distances in the protein in real time. These measurements are based on fluorescence resonance energy transfer (FRET), which reports the molecular proximity of a donor and acceptor fluorophore. We used these approaches to report dynamic changes in distance between the channel and the membrane, between channel subunits, and within a channel subunit. We have recently used transition metal ions at FRET acceptors to develop a breakthrough method that is able to measure the dynamics of the short-range interactions visible in the crystal structure. This has allowed us to observe, for the first time, the rearrangements in the C-helix associated with cyclic nucleotide binding.
From this combination of x-ray crystallography, electrophysiology, and fluorescence measurements, we are beginning to understand how cyclic nucleotide binding to an intracellular domain causes opening of the channel gate in the membrane. Once the cyclic nucleotide is bound, the trigger for channel activation is the movement of the C-helix. This, in turn, causes a rearrangement of the neighboring helices, and disruption of intersubunit interactions in the C-linker. Disrupting the intersubunit interactions releases the hold the C-linker has on the pore gate, allowing it to open. A similar mechanism is probably occurring in all of the members of the cyclic nucleotide–regulated family of channels.