Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

Liu, Yi; Jurman, Mark E.; Yellen, Gary

doi:10.1016/s0896-6273(00)80106-3

Cited by 429 publications

(452 citation statements)

References 38 publications

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“…Consistent with this interpretation, MTSEA reduced ␥ more rapidly when applied directly onto the intracellular aspect of an inside-out patch. Using this approach, the rate of reduction of the amplitude of unitary events was still substantially slower than the rate of MTS modification of simple thiol compounds and easily accessible substituted Cys residues in the outer mouth of the Shaker B K ϩ channel (16,17). Such data suggest that MTSEA encounters a rate-limiting barrier en route to the MA 0Ј residue within the portal.…”

Section: Discussionmentioning

confidence: 94%

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Deeb¹,

Carland

Cooper

et al. 2007

Journal of Biological Chemistry

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Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates ␥, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.Cysteine loop (Cys loop) receptors encompass five families of gene products. Nicotinic-acetylcholine (nACh) 3 receptors, 5-hydroxytryptamine type-3 (5-HT 3 ) receptors, and Zn 2ϩ -activated channels conduct cations, whereas ␥-aminobutyric acid type A and glycine receptors conduct anions (1). Cys loop receptor subunits combine as pentamers forming a central ion pore that traverses the plasma membrane. Studies examining the role of residues comprising the pore-lining second transmembrane (TM2) helices of the nACh receptor led to the traditional view that this region of Cys loop receptors serves as the rate-limiting barrier to ion flux. Among the cationselective Cys loop receptor subunits, conserved acidic residues influence unitary conductance (␥) by forming concentric rings of negative charge positioned along the vertical axis of the central pore (2).Although the role of residues within the TM2 helix of Cys loop receptors in controlling ␥ is well established, recent studies demonstrate a similar function for residues located in the large cytoplasmic TM3-4 loop (1, 3, 4). Based on a 4.6 Å resolution cryoelectron microscopy image of the Torpedo marmorata nACh receptor, Miyazawa et al. (5) first speculated that amino acids in the TM3-4 loop form "transverse tunnels" that may contribute to the ion conduction pathway. Subsequent electrophysiological studies demonstrated that cytoplasmic residues within membrane-associated (MA) helices of the 5-HT 3 and ␣ 4 ␤ 2 nACh receptors are determinants of ␥ (3, 4). Arg 436 in the 5-HT 3A subunit at a position termed MA 0Ј is critical for maintaining the anomalously low ␥ (ϳ900 femtosiemens) that is a hallmark of the homomeric 5-HT 3A receptor. Furthermore, introduction of MA 0Ј Arg into ␣ 4 ␤ 2 nACh receptors halved ␥ (4).A 4 Å resolution model of the T. marmorata nACh receptor indicates that adjacent MA helices frame portals with apertures only slightly larger than partially hydrated permeant cations (6). Using this structure as a template, homology models of the 5-HT 3A and ␣ 4 ␤ 2 nACh receptors indicate that charged amino acids line their cytoplasmic portals (4).Although it is now clear that specific MA helix residues influence the ␥ of nACh and 5-HT 3 receptors, it remains to be determined whether their charge plays a role. We used substituted cysteine modification to address this question, a method fre-

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Section: Discussionmentioning

confidence: 94%

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Deeb¹,

Carland

Cooper

et al. 2007

Journal of Biological Chemistry

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“…Collapse of the permeation pathway at the outer mouth of the pore by motion of the S6 segment gives rise to C-type inactivation. 12 Voltage-gated Na ϩ channels (Nav) and Ca 2ϩ channels (Cav) share sequence similarity and conserved structural features with Shaker K ϩ channels, except that…”

Section: Voltage-gated Ion Channelsmentioning

confidence: 99%

Physiologic Principles Underlying Ion Channelopathies

Cannon

2007

Neurotherapeutics

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Summary:The ion channelopathies are a diverse array of human disorders caused by mutations in genes coding for ion channels. More than 40 different channelopathies have been identified, with representative disorders from every major class of ion channel and affecting all electrically excitable tissues: brain, peripheral nerve, skeletal muscle, smooth muscle, and heart. This review provides an overview of ion channel classification, structure, and function as a framework for understanding which ion channel properties are altered by disease-associated mutations and how these changes disrupt cellular excitability for channelopathies affecting skeletal muscle and the CNS.

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“…This approach allows determination of the residueresidue contacts within the three-dimensional context of a protein (22) and also allows structural changes underlying the activation of a protein to be elucidated (23). The approach involves the introduction of pairs of cysteine residues at positions that are thought to lie in close proximity and then investigating which cysteine pairs can be induced to form a disulfide bridge.…”

mentioning

confidence: 99%

“…The approach involves the introduction of pairs of cysteine residues at positions that are thought to lie in close proximity and then investigating which cysteine pairs can be induced to form a disulfide bridge. The formation of disulfide bridges will often impair or alter the course of further motions, thereby producing a change in the functional properties of the protein (23). In the absence of measurable functional changes, however, disulfides can be detected biochemically (22).…”

mentioning

confidence: 99%

Depolarization Induces Intersubunit Cross-linking in a S4 Cysteine Mutant of the Shaker Potassium Channel

Aziz¹,

Partridge²,

Munsey

et al. 2002

Journal of Biological Chemistry

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Voltage-gated potassium (K v ) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.

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Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

Cited by 429 publications

References 38 publications

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Physiologic Principles Underlying Ion Channelopathies

Depolarization Induces Intersubunit Cross-linking in a S4 Cysteine Mutant of the Shaker Potassium Channel

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