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Summary: William Zagotta is interested in understanding the molecular mechanisms for the function of ion channels.

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 recently 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.

	X-ray crystallographic structure...

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, 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 site-directed mutagenesis, electrophysiological recording, 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 that 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.

At the center of the 4-fold symmetric structure is a tunnel that runs perpendicular to the membrane. The narrowest part of the tunnel is approximately 10 Å in diameter and is lined by a ring of negatively charged amino acids: D487, E488, and D489. Many ion channels have "charge rings" that focus permeant ions at the mouth of the pore and increase channel conductance. We used nonstationary fluctuation analysis and single-channel recording, coupled with site-directed mutagenesis and cysteine modification, to determine whether this part of HCN and CNG channels might be an extension of the permeation pathway. Our results indicate that modifying charge-ring amino acids affects gating but not ion permeation in HCN2 and CNG channels. Thus, this portion of the channel is not an obligatory part of the ion path but instead acts as a "gating ring." The carboxyl-terminal region of these channels must hang below the pore much like the "hanging gondola" of voltage-gated potassium channels, but the permeation pathway must exit the protein before the level of the ring of charged amino acids. (A grant from the National Eye Institute provided support for these experiments.)

The structure of the HCN2 carboxyl-terminal region contains intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. By mutating these salt bridges, we showed that they are present in both the intact HCN2 and CNGA1 channels. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 carboxyl-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. (A grant from the National Eye Institute provided support for these experiments.)

Native rod CNG channels are heterotetramers composed of both CNGA1 and CNGB1 subunits. Many of the specializations of these channels for phototransduction come about from their heteromeric composition. We used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channels in the surface membrane. We found, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. Using a more extensive fluorescence approach, we also showed that the olfactory CNG channel contains CNGA2, CNGA4, and CNGB1b subunits in a ratio of 2:1:1. These results have many implications for CNG channel function in particular and assembly of membrane proteins in general. (A grant from the National Eye Institute provided support for these experiments.)

Ca²⁺/calmodulin binds to a site in the amino-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1/CNGB1 channels. We found that polypeptides derived from an amino-terminal region of CNGB1 form a specific interaction with polypeptides derived from a carboxyl-terminal region of CNGA1 that is distal to the CNBD. Deletion of the Ca²⁺/calmodulin-binding site from the amino-terminal region of CNGB1 eliminated both Ca²⁺/calmodulin modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca²⁺/calmodulin. These results suggest that Ca²⁺/calmodulin-dependent inhibition of rod channels is due to the direct binding of Ca²⁺/calmodulin to a site in the amino-terminal region in CNGB1, which disrupts the interaction between this region and a distal carboxyl-terminal region of CNGA1. More recently we have used simultaneous electrophysiological recording and site-specific fluorescence measurements to confirm this mechanism in intact functional channels in the surface membrane. (A grant from the National Eye Institute provided support for these experiments.)

Last updated: April 4, 2006