Conformational Switch between Slow and Fast Gating Modes

Schönherr, Roland; Mannuzzu, Lidia M.; Isacoff, Ehud Y.; Heinemann, Stefan H.

doi:10.1016/s0896-6273(02)00869-3

Cited by 60 publications

(32 citation statements)

References 59 publications

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“…Such long times are inconsistent with experiment, bringing to bear one of the strongest arguments against the lipid-exposed model as presented here. Biotin-avidin experiments on KvAP have been used to suggest that the S4 helix has extensive contact with lipid (8); however, alanine-scanning mutagenesis on both Shaker and EAG has been used to support a more conservative interaction between just the hydrophobic face of S4 and the membrane (28,29). With regard to gating, diffusion through fluid lipid molecules may be faster than diffusion through a gating canal composed entirely of protein, which highlights an attractive property of the lipidexposed model and suggests that we may have overestimated the diffusion coefficient of the translation model above.…”

Section: Gating Charge Transfer: a Large Gating Charge Mandates Outwamentioning

confidence: 99%

A quantitative assessment of models for voltage-dependent gating of ion channels

Grabe

Lecar

Jan

2004

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

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Voltage-gated ion channels open and close, or “gate,” in response to changes in membrane potential. The electric field across the membrane–protein complex exerts forces on charged residues driving the channel into different functional conformations as the membrane potential changes. To act with the greatest sensitivity, charged residues must be positioned at key locations within or near the transmembrane region, which requires desolvating charged groups, a process that can be energetically prohibitive. Although there is good agreement on which residues are involved in this process for voltage-activated potassium channels, several different models of the sensor geometry and gating motions have been proposed. Here we incorporate low-resolution structural information about the channel into a Poisson–Boltzmann calculation to determine solvation barrier energies and gating charge values associated with each model. The principal voltage-sensing helix, S4, is represented explicitly, whereas all other regions are represented as featureless, dielectric media with complex boundaries. From our calculations, we conclude that a pure rotation of the S4 segment within the voltage sensor is incapable of producing the observed gating charge values, although this shortcoming can be partially remedied by first tipping and then minimally translating the S4 helix. Models in which the S4 segment has substantial interaction with the low-dielectric environment of the membrane incur solvation energies of hundreds of k B T , and activation times based on these energies are orders of magnitude slower than experimentally observed.

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Section: Gating Charge Transfer: a Large Gating Charge Mandates Outwamentioning

confidence: 99%

A quantitative assessment of models for voltage-dependent gating of ion channels

Grabe

Lecar

Jan

2004

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

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“…Shaker, bovine eag, and human eag-related gene channels, such measurements have revealed voltage-dependent changes in fluorescent intensity that correlate fairly well with the steady-state and͞or kinetic properties of ionic or gating currents (23,25,(27)(28)(29). In most cases, these optical signals reflect changes in quenching of the fluorescent probe as a result of voltage sensor conformational changes (23).…”

Section: Methodsmentioning

confidence: 88%

Optical detection of rate-determining ion-modulated conformational changes of the ether-à-go-go K ⁺ channel voltage sensor

Bannister

Chanda

Bezanilla

et al. 2005

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

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In voltage-dependent ether-à -go-go (eag) K ؉ channels, the process of activation is modulated by Mg 2؉ and other divalent cations, which bind to a site in the voltage sensor and slow channel opening. Previous analysis of eag ionic and gating currents indicated that Mg 2؉ has a much larger effect on ionic than gating current kinetics. From this, we hypothesized that ion binding modulates voltage sensor conformational changes that are poorly represented in gating current recordings. We have now tested this proposal by using a combined electrophysiological and optical approach. We find that a fluorescent probe attached near S4 in the voltage sensor reports on two phases of the activation process. One component of the optical signal corresponds to the main charge-moving conformational changes of the voltage sensor. This is the phase of activation that is well represented in gating current recordings. Another component of the optical signal reflects voltage sensor conformational changes that occur at more hyperpolarized potentials. These transitions, which are rate-determining for activation and highly modulated by Mg 2؉ , have not been detected in gating current recordings. Our results demonstrate that the eag voltage sensor undergoes conformational changes that have gone undetected in electrical measurements. These transitions account for the time course of eag activation in the presence and absence of extracellular Mg 2؉ .activation ͉ voltage-gated D rosophila ether-à-go-go (eag) is the original member of a subfamily of voltage-dependent K ϩ channels that includes eag, eag-related gene (erg), and eag-like (elk) channels (1, 2). In mammals, these channels are expressed in the brain and heart, where they play key roles in controlling excitability (2-4). For example, human erg (HERG) channels contribute significantly to the repolarization of the cardiac action potential (3). Disruption of HERG activity results in long QT syndrome, a predisposition to cardiac arrhythmia and sudden death (3,5,6).Eag channels comprise four subunits surrounding a central pore for K ϩ permeation (7). Each subunit has six transmembrane segments. The last two transmembrane segments (S5 and S6) and the intervening P loop form the ion selective pore, whereas S1-S4 form the voltage sensor (8, 9). Voltage control of channel activity is primarily due to the S4 segment, which contains multiple positively charged residues (10). In response to changes in transmembrane voltage, charged residues in S4 initiate conformational changes that control whether the pore is open or closed (11, 12). S4 conformational changes can be measured electrically as gating currents (13).Channels in the eag subfamily are subject to a novel form of regulation by extracellular divalent cations (14-16). In eag channels, the process of voltage-dependent activation is modulated directly by binding of divalent cations to a site in the voltage sensor (16,17). Extracellular divalent ions, including Mg 2ϩ , Mn 2ϩ , and Ni 2ϩ , slow activation kinetics (14, 18). The halfmaximal e...

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“…Atomic force microscopy investigations have shown opening and closing of the channel pore (41,51); however, because these images are surface views, they cannot provide an insight into internal protein changes. Perturbation analysis studies from potassium channels have shown that single site amino acid substitutions can be useful in defining inter-helical associations (52). One way to achieve this goal is the isolation of mutant connexons or channels whereby the mutation causes the hexamer or dodecamer to be locked into a permanent open or closed state.…”

Section: Discussionmentioning

confidence: 99%

Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel

Beahm

Oshima

Gaietta

et al. 2006

Journal of Biological Chemistry

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Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the ␣-and ␤-connexin subgroups but not the ␥-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.Intercellular communication is a fundamental feature of all multicellular organisms. Gap junctions are one means by which cells communicate with each other and arise as tissue cells grow and abut each other. The morphologically distinctive cell-cell junctional areas allow the exchange of ions, nutrients, and small metabolites between neighboring cells. Gap junction structures are found throughout vertebrates and invertebrates, although the primary sequences of constituent proteins are different from each other even though electron micrographs and physiological assays indicate similar quaternary structure and functionality. Gap junctions are composed of two oligomeric channel structures called connexons, with each cell supplying one connexon that docks with the other at their extracellular surfaces. The connexin family of proteins has a very conserved protein folding topology with highly conserved transmembrane and extracellular primary sequences, but contains variable regions of the cytoplasmic loop and C terminus that confer the individual physiological properties to each connexin. Each connexon is made up of a cyclic arrangement of six protein monomers, called connexins (abbreviated as Cx 5 plus the molecular mass of the protein as predicted by the amino acid sequence, e.g. ...

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Conformational Switch between Slow and Fast Gating Modes

Cited by 60 publications

References 59 publications

A quantitative assessment of models for voltage-dependent gating of ion channels

A quantitative assessment of models for voltage-dependent gating of ion channels

Optical detection of rate-determining ion-modulated conformational changes of the ether-à-go-go K ⁺ channel voltage sensor

Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel

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Conformational Switch between Slow and Fast Gating Modes

Cited by 60 publications

References 59 publications

A quantitative assessment of models for voltage-dependent gating of ion channels

A quantitative assessment of models for voltage-dependent gating of ion channels

Optical detection of rate-determining ion-modulated conformational changes of the ether-à-go-go K + channel voltage sensor

Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel

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Optical detection of rate-determining ion-modulated conformational changes of the ether-à-go-go K ⁺ channel voltage sensor