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W N Zagotta

Publications and source records attributed to W N Zagotta.

At least 19 recordsLinked to original sources
PubMed2001

Rotational movement during cyclic nucleotide-gated channel opening.

Cyclic nucleotide-gated (CNG) channels are crucial components of visual, olfactory and gustatory signalling pathways. They open in response to direct binding of intracellular cyclic nucleotides and thus contribute to cellular control of both the membrane potential and intracellular Ca2+ levels. Cytosolic Ni2+ potentiates the rod channel (CNG1) response to cyclic nucleotides and inhibits the olfactory channel (CNG2) response. Modulation is due to coordination of Ni2+ by channel-specific histidines in the C-linker, between the S6 transmembrane segment and the cyclic nucleotide-binding domain. Here we report, using a histidine scan of the initial C-linker of the CNG1 channel, stripes of sites producing Ni2+ potentiation or Ni2+ inhibition, separated by 50 degrees on an alpha-helix. These results suggest a model for channel gating where rotation of the post-S6 region around the channel's central axis realigns the Ni2+-coordinating residues of multiple subunits. This rotation probably initiates movement of the S6 and pore opening.

Amino Acid Sequence↗
PubMed2001

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.

Amino Acid Sequence↗
PubMed2000

Structural basis for ligand selectivity of heteromeric olfactory cyclic nucleotide-gated channels.

In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels.

Animals↗
PubMed2000

Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry.

Site-specific fluorescence recordings have shown great promise in understanding conformational changes in signaling proteins. The reported applications on ion channels have been limited to extracellular sites in whole oocyte preparations. We are now able to directly monitor gating movements of the intracellular domains of cyclic nucleotide-gated channels using simultaneous site-specific fluorescence recording and patchclamp current recording from inside-out patches. Fluorescence signals were reliably observed when fluorophore was covalently attached to a site between the cyclic nucleotide-binding domain and the pore. While iodide, an anionic quencher, has a higher quenching efficiency in the channel's closed state, thallium ion, a cationic quencher, has a higher quenching efficiency in the open state. The state and charge dependence of quenching suggests movements of charged or dipolar residues near the fluorophore during CNG channel activation.

Animals↗
PubMed1999

Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels are activated in response to the direct binding of cyclic nucleotides to an intracellular domain. This domain is thought to contain a beta roll and two alpha helices, designated the B and C helices. To probe the conformational changes occurring in the ligand-binding domain during channel activation, we used the substituted cysteine accessibility method (SCAM). We found that a residue in the beta roll, C505, is more accessible in unliganded channels than in liganded channels, whereas a residue in the C helix, G597C, is more accessible in closed channels than in open channels. These results support a molecular mechanism for channel activation in which the ligand initially binds to the beta roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the beta roll.

Allosteric Site↗
PubMed1999

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels.

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.

Algorithms↗
PubMed1999

Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels.

Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.

Algorithms↗
PubMed1998

Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels.

Native cylic nucleotide-gated (CNG) channels are composed of alpha and beta subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the alpha and beta subunits. Dimers contained three subunit types: alphawt, betawt, and alpham. The alpham subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of alphawtalphawt dimers and from alphawt monomers were indistinguishable. Channels from injection of alphawtalpham dimers had apparent affinities 3-fold lower than alphawt homomultimers, suggesting a channel with two alphawt and two alpham subunits. Channels from coinjection of alphawtalphawt and betabeta dimers were indistinguishable from those composed of alpha and beta monomers and shared all of the characteristics of the alpha+beta phenotype of heteromeric channels. Coinjection of alphawtalpham and beta beta dimers yielded channels also of the alpha+beta phenotype but with an apparent affinity 3-fold lower, indicating the presence of alpham in the tetramer and that alpha+beta channels have adjacent alpha-subunits. To distinguish between an alpha-alpha-alpha-beta and an alpha-alpha-beta-beta arrangement, we compared apparent affinities for channels from coinjection of alphawtalphawt and betaalphawt or alphawtalphawt and betaalpham dimers. These channels were indistinguishable. To further argue against an alpha-alpha-alpha-beta arrangement, we quantitatively compared dose-response data for channels from coinjection of alphawtalpham and beta beta dimers to those from alpha and beta monomers. Taken together, our results are most consistent with an alpha-alpha-beta-beta arrangement for the heteromeric olfactory CNG channel.

Amino Acid Sequence↗
PubMed1998

Kinetic analysis of block of open sodium channels by a peptide containing the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate.

We analyzed the kinetics of interaction between the peptide KIFMK, containing the isoleucine, phen-ylalanine, and methionine (IFM) motif from the inactivation gate, and the brain type IIA sodium channels with a mutation that disrupts inactivation (F1489Q). The on-rate constant was concentration dependent, consistent with a bimolecular reaction with open sodium channels, while the off rates were unaffected by changes in the KIFMK concentration. The apparent Kd was approximately 33 microM at 0 mV. The on rates were voltage dependent, supporting the hypothesis that one or both of the charges in KIFMK enter the membrane electric field. The voltage dependence of block was consistent with the equivalent movement of approximately 0.6 electronic charges across the membrane. In contrast, the off rates were voltage independent. The results are consistent with the hypothesis that the KIFMK peptide enters the pore of the open sodium channel from the intracellular side and blocks it.

Animals↗
PubMed1997

Interdomain interactions underlying activation of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels are multimeric proteins that activate in response to the binding of cyclic nucleotide to intracellular domains. Here, an intramolecular protein-protein interaction between the amino-terminal domain and the carboxyl-terminal ligand-binding domain of the rat olfactory CNG channel was shown to exert an autoexcitatory effect on channel activation. Calcium-calmodulin, which modulates CNG channel activity during odorant adaptation, blocked this interaction. A specific deletion within the amino-terminal domain disrupted the interdomain interaction in vitro and altered the gating properties and calmodulin sensitivity of expressed channels. Thus, the amino-terminal domain may promote channel opening by directly interacting with the carboxyl-terminal gating machinery; calmodulin regulates channel activity by targeting this interaction.

Animals↗
PubMed1997

Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels.

We have examined domain interactions in the rod cyclic nucleotide-gated ion channel using both physiological and biochemical interaction assays. We have found an interaction between two regions of the channel distant in primary structure, the amino-terminal region and the carboxyl-terminal region containing the cyclic nucleotide-binding (CNB) domain. The interaction in functional channels was detected by the formation of a disulfide bond between cysteine residues at position 35 in the amino-terminal region and 481 in the carboxyl-terminal region. The disulfide bond resulted in channel potentiation, which was due, in part, to an increase in availability of C481 to modification when the channels were open. This state dependence is likely to underlie previously reported potentiation of cyclic nucleotide-gated channels by sulfhydryl-reactive compounds. Polypeptides derived from the amino-terminal and carboxyl-terminal regions were shown to interact, even under conditions which precluded disulfide bond formation. These data argue for a previously unknown, direct interaction between disparate regions of channel sequence.

Amino Acids↗
PubMed1997

Mechanism of tetracaine block of cyclic nucleotide-gated channels.

Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 microM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective z delta of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.

Anesthetics, Local↗
PubMed1997

Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels.

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide-gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the alpha subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.

Anesthetics, Local↗
PubMed1996

Altered ligand specificity by protonation in the ligand binding domain of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels are the critical mediators between the second messengers of sensory transduction and the cell's membrane potential. The photoreceptor CNG channels are activated by the direct binding of cGMP but can also be activated to a much lesser extent by cAMP. In rod CNG channels expressed in Xenopus oocytes, we demonstrate two types of potentiation by protons. One type potentiated cGMP-bound and cAMP-bound channels to the same extent, while another potentiated only cAMP-bound channels. Both types of potentiation could be described by a mechanism in which protons bound primarily to the channel open configuration. The potentiation specific to cAMP-bound channels could be accounted for by protonation of aspartic acid 604 (D604). It is the unfavorable electrostatic interaction between the carboxylate of D604 and the purine ring of cAMP that accounts for the normally poor activation of the channels by cAMP. Protonation at this site removed the unfavorable interaction and allowed cAMP to act as nearly a full agonist. Protonation of a second amino acid, H468, contributed to the nucleotide-nonspecific potentiation and is likely to be an element of the channel gating assembly. Protons potentiate native rod channels less than channels formed from subunit 1. In heteromultimeric channels formed by coexpressing subunit 1 with subunit 2, we found a similar attenuation of potentiation. The absence of protonatable amino acids in subunit 2 at positions corresponding to H468 and D604 can explain the reduced effects of pH on native channels.

Animals↗
PubMed1996

Molecular mechanisms of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels are highly specialized to carry out their unique role in cell signalling. Significant progress has been made in the last several years determining the molecular mechanisms for these specializations. The activation of the channels begins with the binding of cyclic nucleotide to a domain in the carboxyl terminal region. This binding, in turn, produces an induced fit of the protein that involves a movement of the C-helix portion of the binding domain. The induced fit of the binding domain is coupled to an allosteric conformational change that opens the channel pore. The pore is formed primarily from the sequence between the S5 and S6 segments. A single glutamic acid in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations, and the site for the effect of protons on permeation.

Amino Acid Sequence↗