Yeast K⁺ Channel

Our patch-clamp survey of the plasma membrane of budding yeast, S. cerevisiae, revealed the activities of one K⁺ channel (1) (Fig. 1) and one mechanosensitive channel (2).

Fig. 1  (a)Methods.  A diagram showing the removal of the yeast cell wall in a hypotonic medium releases the spheroplast that can be patch-clamped.  (b) Burst of unitary current can be registered from a patch of spheroplast membrane (1).  The channel was later identified from the genome sequence as TOK1 (or YKC1).

Upon the completion of the yeast genome sequencing, four groups independently recognized a K⁺- channel gene, TOK1 (YKC1), predicting a subunit with a S₁ S₂ S₃ S₄ S₅ P₁ S₆ S₇ P₂ S₈ transmembrane topology (3,4). 

An unusual property of this two pore-domain K⁺ channel is that it rectifies outwardly, not according to voltage, but to the total electrochemical potential of K⁺ (3,4).  One study viewed TOK1 as an animal inward rectifier inserted backward, but the reported blockage by external Mg²⁺ (3) cannot be replicated.  A detailed kinetic analysis showed that this rectification is due to an intrinsic gating property of the channel filter, which likely collapses when the K⁺ driving force is inward and resurrects when outward (5) (Fig. 2).

Fig. 2  A model of TOK1 gating.  Depicted is the conventional inner gate (IG) defined by the post-pore region and the filter as a second gate (FG).  From genetic and kinetic data, the distinguishable open state(O), rectifying state (R), inter-burst state (IB), and two conventional closed states (C’s) best fit the various positioning of the pore helices (P1 and P2) and the post-pore helices (S6 and S8).  See (7) for detail.

Besides the extremely rapid filter restructuring (akin to C-inactivation), TOK1 also exhibits a set of closed states of conventional kinetics.  Mutations that hamper growth when expressed remove these closed states.  These “gain-of-function” mutations, selected after random mutageneses, were found to be near the cytoplasmic end of S₆ ad S₈ (6) (Fig. 3). These “post-pore” regions, identified by these mutations that block gate closure coincide with the gating peptide and the gating hinge later resolved in the crystal structures of prokaryotic K⁺ channels (kcsA, MthK) by MacKinnon and coworkers.

Fig. 3  “Loose-cannon” TOK1 channel mutations identified the gate.  After a random mutagenesis, TOK1 mutants that hamper growth when expressed were isolated.  Mutant channels examined in Xenopus oocytes were found to lack the closed states (6).  The mutations were found to be located at the “postpore” regions (PP1 and PP2) at the end of S6 and S8.  This genetically defined gate coincides with the one observed in the crystal structures of KcsA and MthK, identifying the conserved G (A) as the gating hinge, a second conserved G at the very constriction, and the gate peptide, as marked.  (Modified from (6).)    See Fig. 1 of “Genetics” of this website for methods.

TOK1 has an additional feature:  the C-terminal cytoplasmic domain of the subunit function to maintain the gate’s open state (7) (Fig. 2).  Surprisingly, this “foot-in-the-door” can be executed in trans, i.e. when the C-terminal domain is expressed as a separate peptide (8). 

1. Gustin et al. (1986) Science 233: 1195.
2. Gustin et al. (1988) Science242: 762.
3. Ketchum et al. (1995) Nature376: 690.
4. Zhou et al. (1995) FEBS lett. 373:170.
5. Loukin & Saimi (1999) Biophy. J.77: 3060.
6. Loukin et al. (1997) EMBO J.16: 4817.
7. Loukin & Saimi (2002) Biophys. J.  82: 781.
8. Loukin et al. (2002) PNAS.99: 1926.

 

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