Conformational Changes in S6 Coupled to the Opening of Cyclic Nucleotide-Gated Channels

Flynn, Galen E.; Zagotta, William N.

doi:10.1016/s0896-6273(01)00324-5

Cited by 157 publications

(158 citation statements)

References 42 publications

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“…It should also be noted that the closed-state modification rates at the three BK pore-lining sites are approximately two orders higher than that of the Shaker channels (23). These rates are even several fold higher than that of closed cyclic nucleotide-gated channels (38), the ion perme- ation gate of which has been determined to be in the selectivity filter region (38,39). In accord with previous results that small molecules such as quaternary ammonium blockers may pass in and out of the BK inner pore in both open and closed states (17,20), this observation suggests that any constriction at the cytosolic end of BK S6 does not prevent access of molecules to the inner pore and thus is unlikely to form an ion permeation gate.…”

Section: Discussionmentioning

confidence: 97%

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

Zhou

Xia

Lingle

2011

Proc. Natl. Acad. Sci. U.S.A.

105

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BK channels are regulated by two distinct physiological signals, transmembrane potential and intracellular Ca 2+ , each acting through independent modular sensor domains. However, despite a presumably central role in the coupling of sensor activation to channel gating, the pore-lining S6 transmembrane segment has not been systematically studied. Here, cysteine substitution and modification studies of the BK S6 point to substantial differences between BK and Kv channels in the structure and function of the S6-lined inner pore. Gating shifts caused by introduction of cysteines define a pattern and direction of free energy changes in BK S6 distinct from Shaker. Modification of BK S6 residues identifies pore-facing residues that occur at different linear positions along aligned BK and Kv S6 segments. Periodicity analysis suggests that one factor contributing to these differences may be a disruption of the BK S6 α-helix from the unique diglycine motif at the position of the Kv hinge glycine. State-dependent MTS accessibility reveals that, even in closed states, modification can occur. Furthermore, the inner pore of BK channels is much larger than that of K + channels with solved crystal structures. The results suggest caution in the use of Kv channel structures as templates for BK homology models, at least in the pore-gate domain.cysteine modification | K channels | Slo1 channels A s a unique member of the family of K + channels, large conductance, Ca 2+ -activated K + (BK or Slo1) channels are activated in a highly synergistic manner by two distinct physiological signals: depolarization and intracellular Ca 2+ . To accomplish this, each of the four α-subunits in a functional BK channel is constructed of three modular parts: a voltage-sensing domain composed of S1 to S4 transmembrane segments, a huge cytosolic domain sensing various intracellular ligands, such as Ca 2+ (1, 2), Mg 2+ (3), and heme (4, 5), and a pore-gate domain (PGD) formed by S5-pore loop-S6 to allow selective K + permeation under the regulation of transmembrane potential and [Ca 2+ ] i (6-8).The BK channel shares homology in its membrane-associated domain, including the voltage-sensing domain and PGD, with voltage-dependent K + (Kv) channels. Thus, the X-ray crystallographic structures of Kv channels (9, 10) have been widely used as an important template to guide structure-function studies of BK channels (11-13). However, the distant evolutionary relationship between BK and Kv channels (14) raises the possibility that Kv channel structure must be used with caution as a guide to BK channel structure. Sequence alignment (Fig. 1A) shows that BK channels and Kv channels differ at two critical positions in their pore-lining S6 transmembrane segments. First, there are two consecutive glycines (i.e., diglycine) at the conserved glycine hinge (15) in BK channels, but only one in the corresponding region of Kv channels. Second, whereas Kv channels share a conserved PXP motif near the cytosolic entrance to the inner pore, in BK channels there is only one prol...

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Section: Discussionmentioning

confidence: 97%

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

Zhou

Xia

Lingle

2011

Proc. Natl. Acad. Sci. U.S.A.

105

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“…For example, studies using intracellularly applied molecules that block the permeation pathway of CNG channels, such as divalent ions (18), tetracaine (19,20), or quaternary ammonium ions (21), have shown that blockade is not state-dependent, as if these molecules can access the pore in both open and closed channels, which is in stark contrast with the blockade properties observed in K V channels (10,11,14,(22)(23)(24). In addition, experiments examining the state dependence of cysteine modification by intracellular application of methanethiosulfonate (MTS) reagents have failed to show dramatic differences between open and closed states in the inner-vestibule region (21,25,26), results that are inconsistent with an intracellular gate in TM6, as shown in K V channels (12,15).Several studies indicate that the pore region of CNG channels plays a role in gating. For example, accessibility of cysteine reagents applied from the intracellular (27, 28) and the extracellular (27-29) side of the channel to cysteines substituted along the entire P region have shown that modification of some residues in this region perturbed normal gating by cGMP.…”

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confidence: 96%

Gating at the selectivity filter in cyclic nucleotide-gated channels

Contreras

Srikumar²,

Holmgren³

2008

Proc. Natl. Acad. Sci. U.S.A.

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By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (KV) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.cGMP ͉ ion channel ͉ signal transduction C yclic nucleotide-gated (CNG) channels sense variations in the intracellular concentration of cyclic nucleotides that occur in response to visual or olfactory stimuli, therefore playing essential roles in the transduction of visual and olfactory information (1, 2). In many ways, CNG channels are similar to voltage-activated potassium (K V ) channels. They coassemble as tetramers of homologous subunits (3-6), each containing six transmembrane segments (TM), a positively charged TM4 and a reentry P region between TM5 and TM6, suggesting that CNG channels belong to the same superfamily of voltage-activated cation channels (7). The main difference is that CNG channels are only weakly voltage-dependent. Instead, they open and close the pore in response to changes in the intracellular concentrations of cGMP or cAMP, a property conferred by the presence of a cyclic nucleotide binding domain at the C terminus of each subunit (8, 9).Our understanding of how CNG channels open and close their pore in response to cyclic nucleotide binding is much less refined than our understanding of how K V channels gate in response to voltage. A large body of evidence, using a variety of approaches, has established that K V channels open and close their permeation pathway at the intracellular end of the pore (10-17). Attempts to extend those ideas to CNG channels have encountered some resistance. For example, studies using intracellularly applied molecules that block the permeation pathway of CNG channels, such as divalent ions (18), tetracaine (19,20), or quaternary ammonium ions (21), have shown that blockade is not state-dependent, as if these molecules can access the pore in both open and closed channels, which is in stark contrast with the blockade properties observed in K V channels (10,11,14,(22)(23)(24). In addition, experiments examining the state dependence of cysteine modification by intracellular application of methanethiosulfonate (MTS) reagents have failed to show dramatic differences between open and closed states in the inner-vestibule region (21,25,26), results that are inconsistent with an intracellular gate in TM6, as shown in K V channels (12,15).Se...

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“…For many ion channel proteins, the portion of the protein that detects the activating signal (46 -50) or acts as the activation gate (51-54) may be as large as an entire TM domain consisting of several turns of an ␣-helix (55,56). The gate of Shaker-type channels has been studied by comparing atomic structures of the closed state of the KcsA channel with the open state of the calciumactivated MthK channel (57,58).…”

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confidence: 99%

The Gate of the Influenza Virus M2 Proton Channel Is Formed by a Single Tryptophan Residue

Tang¹,

Zaitseva²,

Lamb

et al. 2002

Journal of Biological Chemistry

234

319

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The influenza virus M 2 proton-selective ion channel is known to be essential for acidifying the interior of virions during virus uncoating in the lumen of endosomes. The M 2 protein is a homotetramer that contains four 19-residue transmembrane (TM) domains. These TM domains are multifunctional, because they contain the channel pore and also anchor the protein in membranes. The M 2 protein is gated by pH, and thus we have measured pH-gated currents, the accessibility of the pore to Cu 2؉ , and the effect of a protein-modifying reagent for a series of TM domain mutant M 2 proteins. The results indicate that gating of the M 2 ion channel is governed by a single side chain at residue 41 of the TM domain and that this property is mediated by an indole moiety. Unlike many ion channels where the gate is formed by a whole segment of a protein, our data suggest a model of striking simplicity for the M 2 ion channel protein, with the side chain of Trp 41 blocking the pore of the M 2 channel when pH out is high and with this side chain leaving the pore when pH out is low. Thus, the Trp 41 side chain acts as the gate that opens and closes the pore.The prediction that the influenza A virus M 2 protein has a proton-selective ion channel activity (Refs. 1 and 2 and reviewed in Ref.3) arose from a coupling of various observations on the life cycle of influenza virus. The M 2 protein is an integral membrane protein that is expressed at the plasma membrane of influenza virus-infected cells and is incorporated in small amounts into budding virions (4, 5). Studies on the mechanism of action of the anti-viral drug, amantadine (1-aminoadamantine hydrochloride), indicated that viral escape mutants resistant to the drug mapped to the transmembrane (TM) 1 domain of the M 2 protein (6) and that amantadine affected two steps in the life cycle, virus uncoating and virus maturation. The effect of amantadine on inhibition of uncoating is general to all strains of influenza A virus (7, 8) (reviewed in Refs. 3, 9 -11). When a virion has entered the cell by receptor-mediated endocytosis and the virus particle is in the acidic environment of the endosomal lumen, the M 2 ion channel is activated and conducts protons across the viral membrane. The lowered internal virion pH is thought to weaken protein-protein interactions between the viral matrix protein (M1) and the ribonucleoprotein (RNP) core (7,(12)(13)(14)(15)). In the presence of amantadine, influenza virus uncoating is incomplete, because the M1 protein is not released from the RNPs and the RNPs fail to enter the nucleus. Normally, influenza virus RNPs are transcribed and replicated in the nucleus (reviewed in Ref. 10). For some influenza virus subtypes, amantadine inhibits a "late" step in virus replication. The M 2 ion channel activity is activated during transport of the M 2 protein through the exocytic pathway; this ion channel activity raises the lumenal pH of the trans Golgi network (TGN), equilibrating pH with that of the cytoplasm (1, 17-22). Thus, the intralumenal pH of the TGN i...

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Conformational Changes in S6 Coupled to the Opening of Cyclic Nucleotide-Gated Channels

Cited by 157 publications

References 42 publications

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

Gating at the selectivity filter in cyclic nucleotide-gated channels

The Gate of the Influenza Virus M2 Proton Channel Is Formed by a Single Tryptophan Residue

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