Physiological Characterization of the Yeast Plasma Membrane Outward Rectifying K + Channel, DUK1 (TOK1), In Situ

Bertl, Adam; Bihler, Hermann; Reid, John D.; Kettner, Carsten; Slayman, Clifford L.

doi:10.1007/s002329900343

Cited by 57 publications

(73 citation statements)

References 63 publications

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“…This result could indicate either that phosphorylation in fact has no role in TOK1 gating, that the dephosphorylated form is the functional form, or that the phosphatase failed to dephosphorylate the channel. Consistent with the notion that cytoplasmic-side phosphorylation is important, TOK1 channel rundown could result from loss of tail function and can be prevented when ATP is added to the bath of membrane patches excised from yeast (29).…”

Section: Discussionsupporting

confidence: 69%

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K ⁺ channel

Loukin

Lin

Athar

et al. 2002

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

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Non-targeted mutagenesis studies of the yeast K ؉ channel, TOK1, have led to identification of functional domains common to other cation channels as well as those so far not found in other channels. Among the latter is the ability of the carboxyl tail to prevent channel closure. Here, we show that the tail can fulfill this function in trans. Coexpression of the carboxyl tail with the tail-deleted channel core restores normal channel behavior . A Ser͞Thr-rich region at its amino end and an acidic stretch at its carboxyl end delineate the minimal region required for tail function. This region of 160 aa apparently forms a discrete functional domain. Interaction of this domain with the channel core is strong, being recalcitrant to removal from excised membrane patches by both high salt and reducing agents. Although the use of a cytoplasmic domain to regulate channel is common among animal channels, by using it as a ''foot-in-the-door'' to maintain open state appears unique to TOK1, the first fungal K ؉ channel studied in depth.Saccharomyces cerevisiae ͉ TOK1 ͉ channel gating T he K ϩ -specific ion channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae (TOK1) is one of the first microbial K ϩ channels to be examined in detail. Genetic analysis, coupled with biophysical analysis, of TOK1 has assisted in the identification of what we call the PP region, the cytoplasmic end of the P-region-following membrane domain, which has been found to be intimately involved with gating of cation channels (1, 2, 3). Our analyses have also pointed to the existence of functional domains that, on first glance, appeared unique to TOK1, including a filter-specific gating (Fig. 1A; ref. 22) and a carboxyl tail stabilization of the inner-pore gate (Fig. 1B; ref. 4). Further characterization of these ''unique'' properties are important not just for the sake of understanding TOK1 function, but for possible general relevance. Both filter-specific gating (5-8) and cytoplasmic domain influence of channel gating (9-18) are emerging as common themes amongst K ϩ channels. Here, we report on a more specific analysis of the ability of the carboxyl tail to influence TOK1 gating.TOK1 is predicted to contain eight transmembrane domains with canonical P-region pore loops following both the fifth and seventh spans (19,20). The biophysics and genetics of this channel have been explored, but its physiological function remains largely unknown except as a target for killer toxin (6, 7). Although commonly referred to as ''voltage-regulated'' (21), TOK1's gating is regulated by the K ϩ electrochemical potential (⌬ K ϩ ) rather than merely voltage alone (19,20). The front line of this ⌬ K ϩ -dependent regulation is a nearinstantaneous gating process that prevents inward current flow, the ''R'' state (1). Because it is the only place where the transmembrane ⌬ K ϩ can be efficiently monitored, the R state is most readily accounted for as an intrinsic gating property of the filter region (22). We proposed a model in which inward ⌬ K ϩ results...

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Section: Discussionsupporting

confidence: 69%

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K ⁺ channel

Loukin

Lin

Athar

et al. 2002

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

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“…We have recently succeeded in whole cell patch clamp recordings of the yeast giant protoplasts as well. Thus, a combination of these two methods together shall provide a powerful system for analyzing any ion transporter, not only highly active channels (5)(6)(7)(8)(9)(10)(11)(12)(45)(46)(47)(48) but also other weaker transporters in the membranes, either vacuolar or cytoplasmic, of S. cerevisiae. We used haploid cells because they are much easier than polyploid ones to manipulate genes to yield deletion mutants.…”

Section: H ϩ -Pump Current Of V-type Atpase In Yeast Giant Vacuolesmentioning

confidence: 99%

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae

Yabe¹,

Horiuchi²,

Nakahara³

et al. 1999

Journal of Biological Chemistry

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Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(⌬vma1::TRP)). The K m value for the ATP dose response of the current was 159 M and the H ؉ /ATP ratio estimated from the reversible potential of the V-I curve was 3.5 ؎ 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

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“…The channel activity was strongly voltage dependent (with a high value of the apparent gating charge), which indicated that the amplitudes of the steady-state whole-cell currents were strongly outwardly rectified. It is considered that the timeaveraged current-voltage relationship qualitatively corresponds to the I/V relationship of the steady-state whole-cell currents (2,4). Therefore, in order to show rectification, we plotted against the voltage ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Outwardly Rectifying Anionic Channel from the Plasma Membrane of the Fungus Phycomyces blakesleeanus

Živić

Popović²,

Todorović³

et al. 2009

Eukaryot Cell

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In the present report, by using a patch clamp technique, we provide, to our knowledge, the first detailed description of an anionic channel from filamentous fungi. The characterized channel, an outwardly rectifying anionic channel ( The molecular identities and electrophysiological and structural properties of anion-selective channels are well characterized in animal cells and, to a lesser extent, in plant cells (7,22,29,31). In fungi, on the other hand, data on anion channels are very scarce. Up to now, only two electrophysiological studies have demonstrated the existence of anion channels in fungal membranes. Both of these studies, however, were mainly methodological, dealing with the development of techniques for successful patch clamping (i.e., gigaseal formation) of fungal plasma membranes. The channel activity recordings primarily illustrated the validity of the techniques. The available data show that anion channels from cell membranes of Neurospora crassa (37) and Aspergillus niger (33) are selective for Cl Ϫ over K ϩ , with single-channel conductances of 17 pS and 43 pS, respectively, and that the one from N. crassa is a weak outward rectifier while the one from A. niger is a strong rectifier. No data on selectivity among anions, kinetic parameters, or blockers of these channels are available.The lack of knowledge about fungal anion channels is mainly the result of technical difficulties in the formation of a highresistance seal, a"gigaohm seal" (Ͼ1 G⍀) between hyphal plasma membranes and patch clamp pipettes. The "gigaohm seal" is necessary for detailed analysis of ion channel properties (i.e., selectivity and gating). These difficulties are a consequence of the specific structure of the fungal cell wall. The only readily patchable fungal cell membrane is that of a yeast (Saccharomyces cerevisiae), the experimental model used in virtually all electrophysiological characterizations of fungal cationic channels (3). None of these experiments showed anion channel activity, indicating the absence of anionic channels. However, unicellular organization and cell walls composed of mannans and glucans clearly separate yeasts from the majority of other fungi denoted filamentous fungi. Filamentous fungi are characterized by multicelullar mycelia, polarized hyphal growth, and chitin cell walls. Electrophysiological study of the only cationic channel from a filamentous fungus (N. crassa) that has been well characterized was conducted by cloning and expression of the channel in yeast plasma membranes (32).Removal of the cell wall with cell wall-degrading enzymes, which gave good results in plants, failed to solve the problem in fungi, since patch clamping of the obtained protoplasts resulted only in subgigaohm seals that were not suitable for detailed examination of ionic channels (14,25). The only exception is the report of a gigaohm seal on enzyme-derived germling protoplast from Uromyces (40). In another report, the problem of cell wall removal was resolved by using a wall-less slime mutant of N. crassa (24). Desp...

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Physiological Characterization of the Yeast Plasma Membrane Outward Rectifying K + Channel, DUK1 (TOK1), In Situ

Cited by 57 publications

References 63 publications

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K ⁺ channel

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K ⁺ channel

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae

Outwardly Rectifying Anionic Channel from the Plasma Membrane of the Fungus Phycomyces blakesleeanus

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Physiological Characterization of the Yeast Plasma Membrane Outward Rectifying K + Channel, DUK1 (TOK1), In Situ

Cited by 57 publications

References 63 publications

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K + channel

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K + channel

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae

Outwardly Rectifying Anionic Channel from the Plasma Membrane of the Fungus Phycomyces blakesleeanus

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About

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K ⁺ channel

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K ⁺ channel