Constraining Ligand-Binding Site Stoichiometry Suggests that a Cyclic Nucleotide–Gated Channel Is Composed of Two Functional Dimers

Liu, David T.; Tibbs, Gareth R.; Paoletti, Pierre; Siegelbaum, Steven A.

doi:10.1016/s0896-6273(00)80530-9

Cited by 137 publications

(156 citation statements)

References 40 publications

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“…CNG channels from rod photoreceptors and olfactory neurons were shown to consist of three CNGA subunits and one CNGB subunit, similar to the Kv2.1͞Kv9.3 complexes described here (17,42,43). Whereas for CNG channels, considerable evidence for structural and functional organization as dimers of dimers exists (44,45), a C-terminal leucine zipper domain present in CNGA subunits, but not CNGB subunits, supports a trimeric interaction of CNGA subunits, and thus, favors asymmetric tetramerization (42). At present, no such domain is known for Kv2.1 subunits.…”

Section: Discussionmentioning

confidence: 52%

Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Kerschensteiner

Soto

Stocker

2005

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

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Modulatory ␣-subunits, which comprise one-fourth of all voltagegated K ؉ channel (Kv) ␣-subunits, do not assemble into homomeric channels, but selectively associate with delayed rectifier Kv2 subunits to form heteromeric channels of unknown stoichiometry. Their distinct expression patterns and unique functional properties have made these channels candidate molecular correlates for a broad set of native K ؉ currents. Here, we combine FRET and electrophysiological measurements to determine the stoichiometry and geometry of heteromeric channels composed of the delayed rectifier Kv2.1 subunit and the modulatory Kv9.3 ␣-subunit. Kv channel ␣-subunits were fused with GFP variants, and heteromerization of different combinations of tagged and untagged ␣-subunits was studied. FRET, evaluated by acceptor photobleaching, was only observed upon formation of functional channels. Our results, obtained from two independent experimental paradigms, suggest the formation of heteromeric Kv2.1͞Kv9.3 channels of fixed stoichiometry consisting of three Kv2.1 subunits and one Kv9.3 subunit. Strikingly, despite this uneven stoichiometry, we find that heteromeric Kv2.1͞Kv9.3 channels maintain a pseudosymmetric arrangement of subunits around the central pore.FRET ͉ voltage-gated K ϩ channel ͉ potassium channel ͉ silent subunit

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Section: Discussionmentioning

confidence: 52%

Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Kerschensteiner

Soto

Stocker

2005

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

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“…This was done by studying single channels comprised of mixtures of functional and nonfunctional subunits in which the high-affinity allosteric activator was knocked out by mutating the Ca-bowl. The stoichiometry of each channel was determined from the single channel current amplitude by introducing a mutation to a tetraethylammonium (TEA) sensitive site (12,26). Our results show directly that BK channels can open with 0, 1, 2, 3, or 4 functional Ca-bowls, and that each subunit with a functional Ca-bowl contributes a similar stepwise increase to the cooperativity.…”

mentioning

confidence: 80%

“…2. Differences in properties of channels depending on whether altered subunits are adjacent or diagonal have been observed (26,43,44).…”

Section: Discussionmentioning

confidence: 99%

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca ²⁺

Niu

Magleby

2002

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

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BK channels (Slo1) are widely distributed K+ channels that control Ca2+-dependent processes and cellular excitability. Their activation by intracellular Ca2+ (Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}) is highly cooperative, with Hill coefficients of typically 2–5. To investigate the cooperativity contributed by each of the four α subunits that form the BK channel, we studied single channels comprised of mixtures of functional subunits and subunits with a mutation to disrupt a key site (Ca-bowl) required for activation by low concentrations of Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}. As the number of functional subunits increased, we found a stepwise increase in the Hill coefficient of 0.3–0.8 per functional subunit and a stepwise decrease in the Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document} required for half activation (Kd). These results show directly that BK channels can open with 0, 1, 2, 3, or 4 functional Ca-bowls, and that each subunit with a functional Ca-bowl contributes a stepwise increase to both the cooperativity of activation and the apparent Ca2+ affinity. A model with 0–4 high-affinity allosteric activators and four low-affinity allosteric activators was examined. In this model, Ca2+ bindings were independent of one another and the cooperativity arose from the joint action of the allosteric activators on the open–closed equilibrium. Although this model described well the major features of the experimental data, some differences between the observed and predicted results indicated that additional factors not included in the model also contribute to the cooperativity

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“…As such, they are expected to be at a substantial distance from each other, making it difficult to conceive motions that would bring the N-terminal ends together without disrupting the tetrameric symmetry. However, if we view the channel as a symmetric dimer of dimers, as has been proposed previously for K v channels (37) and the cyclic nucleotide gated channels (38,39), and consider the recent report that in SK Ca channels gating occurs through a dimerization of the intracellular regulatory domains (8,40), it would be possible to imagine S4s from neighboring subunits moving closer to each other. Structural data and further protein chemistry experiments are required to test this possibility and propose models of S4 motion.…”

Section: Discussionmentioning

confidence: 96%

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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Constraining Ligand-Binding Site Stoichiometry Suggests that a Cyclic Nucleotide–Gated Channel Is Composed of Two Functional Dimers

Cited by 137 publications

References 40 publications

Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca ²⁺

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

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Constraining Ligand-Binding Site Stoichiometry Suggests that a Cyclic Nucleotide–Gated Channel Is Composed of Two Functional Dimers

Cited by 137 publications

References 40 publications

Fluorescence measurements reveal stoichiometry of K + channels formed by modulatory and delayed rectifier α-subunits

Fluorescence measurements reveal stoichiometry of K + channels formed by modulatory and delayed rectifier α-subunits

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca 2+

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

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Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Fluorescence measurements reveal stoichiometry of K ⁺ channels formed by modulatory and delayed rectifier α-subunits

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca ²⁺