Functional Stoichiometry of Shaker Potassium Channel Inactivation

MacKinnon, Roderick; Aldrich, Richard W.; Lee, Alice W.

doi:10.1126/science.7694359

Cited by 185 publications

(170 citation statements)

References 8 publications

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“…These findings support the "ball and chain" hypothesis of channel inactivation originally proposed to explain rapid inactivation of voltage-gated Na + channels (Bezanilla and Armstrong, 1977). Although voltage-gated K + channels are tetrameric, a single gate seems sufficient to inactivate Shaker K + channels (MacKinnon, 1991;MacKinnon et al, 1993), and basic and hydrophobic amino acids within the N-terminal domain are critical to the interaction of the inactivation gate with the internal mouth of the channel pore (MurrellLagnado andAldrich, 1993a, 1993b).…”

Section: Introductionsupporting

confidence: 82%

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

Covarrubias

Wei

Salkoff

et al. 1994

Neuron

141

124

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SummaryThe effect of protein kinase C (PKC) on rapid N-type inactivation of K + channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K + channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.

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Section: Introductionsupporting

confidence: 82%

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

Covarrubias

Wei

Salkoff

et al. 1994

Neuron

141

124

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“…Given the established photochemistry of the 2-nitrobenzyl group (8)(9)(10)(11)(12)(13)(14)(15)(16)) and the well characterized Shaker-IR phenotype (26)(27)(28)(29)(30)(31)(32)(33)(34)(35), these results provide convincing evidence that irradiation of Npg leads to peptide backbone cleavage of functional ion channels in vivo. Photolysis of the Npg-containing channels resulted in the liberation of the inactivation ball from the remainder of the channel, thereby slowing inactivation.…”

Section: Resultsmentioning

confidence: 69%

“…Photolysis of the Npg-containing channels resulted in the liberation of the inactivation ball from the remainder of the channel, thereby slowing inactivation. Because each channel contains four independent balls, with only a single ball necessary to produce inactivation, the rate and extent of inactivation depend on the number of balls (35). The photolyzed channels are indistinguishable from channels produced in systems expressing an appropriate mixture of ShB and Shaker-IR, and, based on those studies, we estimated that we have removed on average two of the four balls from ShB channels after irradiation for 4 h.…”

Section: Resultsmentioning

confidence: 99%

Site-specific, photochemical proteolysis applied to ion channels in vivo

England

Lester

Davidson

et al. 1997

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

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A method for site-specific, nitrobenzylinduced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K ؉ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the ''signature'' Cys128-Cys142 disulfide loop of the nAChR ␣ subunit.A powerful tool for probing protein structure and function is the incorporation of unnatural amino acids by the method of nonsense suppression, a technique developed for in vitro translation systems by Schultz and coworkers (1-3). Recent work has expanded the scope of this methodology by establishing procedures for unnatural amino acid incorporation into proteins expressed in living cells (4-7). This allows the evaluation of neuroreceptors, ion channels, and related proteins that are studied most effectively in vivo using heterologous expression systems such as the Xenopus oocyte. The suppression methodology is especially promising for such integral membrane proteins, which are not yet generally amenable to the methods of high resolution structure determination (e.g., NMR, x-ray crystallography).In this paper we (i) describe the unnatural amino acid 2-(nitrophenyl)glycine (Npg) and show that it can be efficiently incorporated into proteins expressed in vivo; (ii) establish that in vivo irradiation of an Npg-containing protein leads to site-specific, nitrobenzyl-induced photochemical proteolysis (SNIPP) (Fig. 1); and (iii) illustrate the usefulness of this residue by cleavage of the Cys128-Cys142 ''signature'' disulfide loop of the nicotinic acetylcholine receptor, establishing a crucial functional role for this structural feature.The design of the unnatural amino acid Npg was based on the photochemistry of 2-nitrobenzyl derivatives. Compounds of this type, including Npg itself, have been used as protecting groups in organic synthesis and to produce ''caged'' neurotransmitters, ions, and second messengers that can then be liberated photochemically, but, to our knowledge, Npg has not been incorporated into peptides or proteins (8-17). As shown in Fig. 1, incorporation of Npg into a protein, followed by irradiation of the protein, was anticipated to produce peptide backbone cleavage (18) dures (19-20).-(2-nitrophenyl)glycine was protected as the N-pent-4-enoyl (4PO) derivative using standard conditions (21, 22). Na 2 CO 3 (111 mg, 1.05 mmol) was added to a room temperature solution of (2-nitrophenyl)glycine hydrochloride (82 mg, 0.35 mmol) in H 2 O:dioxane (0.75 ml:0.5 ml), followed by a solution of pent-4-enoic anhydride (70.8 mg, ...

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“…The second point is reinforced by previous work concerning disulfide bridge formation between C51 residues in neighboring TASK2 subunits, where chemical reduction of the disulfide bond by DTT caused dissociation of channel subunits, yet the pH sensitivity was unaltered (13). The redundancy of having two, rather than one, pH-sensing loops is somewhat akin to that of N-type inactivation in voltage-gated channels, in which inactivation requires the presence of only one ball domain, whereas WT channels contain four such domains (14).…”

Section: Network Of Charged Residues In the M1-p1 Loop Of A Single Sumentioning

confidence: 88%

pH sensing in the two-pore domain K ⁺ channel, TASK2

Morton

Abohamed

Sivaprasadarao

et al. 2005

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

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TASK2 is a member of the two-pore domain K ؉ channel family that plays a role in acid-base homeostasis; TASK2 knockout animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis. It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH sensing is not understood. We have examined the role of charged residues in the extracellular domains in pH sensing using a mutational approach. Mutant channels were expressed in CHO cells and studied by whole-cell and single-channel patch clamp. Neutralization of no single amino acid in isolation gave complete loss of pH sensitivity. However, the combined removal of five charged amino acids in the large extracellular loop linking the first transmembrane and pore domains, the M1-P1 loop, resulted in an essentially pH-insensitive channel, stabilized in the open state. Wild-type channels contain two such loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH-sensitive, indicating that only one M1-P1 loop is required to yield a fully pH-sensitive channel, demonstrating a regulatory role of this distinctive structure in two-pore domain K ؉ channels. Thus, pH sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1-P1 loop.potassium channel ͉ regulation ͉ acid ͉ proton ͉ gating

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Functional Stoichiometry of Shaker Potassium Channel Inactivation

Cited by 185 publications

References 8 publications

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

Site-specific, photochemical proteolysis applied to ion channels in vivo

pH sensing in the two-pore domain K ⁺ channel, TASK2

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Functional Stoichiometry of Shaker Potassium Channel Inactivation

Cited by 185 publications

References 8 publications

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

Site-specific, photochemical proteolysis applied to ion channels in vivo

pH sensing in the two-pore domain K + channel, TASK2

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pH sensing in the two-pore domain K ⁺ channel, TASK2