Functional properties of a truncated recombinant GIRK5 potassium channel

In an attempt to understand the relative contribution of each one of the 2P from TOK1 to the functional properties of this channel, we split and expressed the pore domains separately or in combination. Expression of the two domains separately rescued a potassium transport-deficient yeast mutant, suggesting that each domain forms functional potassium-permeable channels in yeast. In Xenopus laevis oocytes expression of each pore domain resulted in the appearance of unique inwardly rectifying cationic channels with novel gating and pharmacological properties. Both pore domains were poorly selective to potassium; however, upon co-expression they partially restored TOK1 channel selectivity. The single channel conductance was different in both pore domains with 7 ؎ 1 (n ‫؍‬ 12) and 15 ؎ 2 (n ‫؍‬ 12) picosiemens for the first and second domain, respectively. In light of the known structure of the Streptomyces lividans KcsA potassium channel pore (see Doyle et al. above), these results suggest a novel non-four-fold-symmetric architecture for 2P potassium-selective channels.Potassium channels play key roles in the physiology of prokaryotic and eukaryotic organisms. In many cells, potassium channels are responsible for maintaining the resting membrane potential and for the modulation of firing properties in excitable cells (4). The exceptionally diverse functional properties of potassium channels are matched by a large number of genes identified over the last few years (5).Genome sequencing and molecular cloning has allowed the identification of a significant number of potassium channels possessing two and six transmembrane domains (TM) 1 (5). Despite the structural diversity, all these channels have a common feature consisting of a single pore-forming domain, which is essential for ion conduction and selectivity (1, 2). It is generally accepted that all these potassium channels aggregate as tetramers to form a functional channel, leaving the pore at the axis of a four-fold symmetry (1, 6).Recently a new family of potassium channels characterized by the presence of two pore-forming domains in tandem (2P) has been identified (3, 7-9). These new potassium channels have either four (2P/4TM) or eight (2P/8TM) transmembrane segments and are highly conserved throughout evolution (7,11,12). Recent experimental evidence suggests that this family of potassium channels probably dimerizes to form functional channels (8). In general, the sequences of both selectivity filters of 2P channels are different, thus they are expected to make potassium-selective pores without the regular four-fold symmetry, but the functional significance of having two different pore-forming domains remains largely unknown.TOK1 was the first member identified from the 2P family of potassium channels (3). This is the only member from this family possessing eight TM (2P/8TM). The arrangement of the putative TM and the 2P in TOK1 results in a structure that resembles a six TM Shaker-like channel attached to an inward rectifier-like channel. This potassium chann...

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“…2). The amplitude of these currents is in agreement with what other groups have observed in X. laevis obtained from different suppliers (18,19).…”

Section: Dividing the Two Pore Domains From Tok1 Produces Novelsupporting

confidence: 92%

Splitting the Two Pore Domains from TOK1 Results in Two Cationic Channels with Novel Functional Properties

Saldaña¹,

Naranjo²,

Coria³

et al. 2002

Journal of Biological Chemistry

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“…Another member of this family is GIRK5, an endogenous G-protein activated potassium channel present only in Xl oocytes [12]. GIRK5 is a homotetramer that helps to maintain a hyperpolarized resting membrane potential; GIRK5 shows a basal activity due to an endogenous G-βγ protein pool that keeps oocytes meiosis arrest [13]–[16]. Interestingly, in contrast to its mammalian homologues, GIRK5 contains a particularly long N-terminus (Fig.…”

Section: Introductionmentioning

confidence: 99%

The Polarization of the G-Protein Activated Potassium Channel GIRK5 to the Vegetal Pole of Xenopus laevis Oocytes Is Driven by a Di-Leucine Motif

et al. 2013

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The G protein-coupled inwardly-rectifying potassium channels (known as GIRK or Kir3) form functional heterotetramers gated by G-βγ subunits. GIRK channels participate in heart rate modulation and neuronal postsynaptic inhibition in mammals. In Xenopus laevis oocytes, GIRK5 is a functional homomultimer. Previously, we found that phosphorylation of a tyrosine (Y16) at its N-terminus downregulates the surface expression of GIRK5. In this work, we elucidated the subcellular localization and trafficking of GIRK5 in oocytes. Several EGFP-GIRK5 chimeras were produced and an ECFP construct was used to identify the endoplasmic reticulum (ER). Whereas GIRK5-WT was retained in the ER at the animal pole, the phospho-null GIRK5-Y16A was localized to the vegetal pole. Interestingly, a construct with an N-terminal Δ25 deletion produced an even distribution of the channel in the whole oocyte. Through an alanine-scan, we identified an acidic cluster/di-leucine sorting-signal recognition motif between E17 and I22. We quantified the effect of each amino acid residue within this di-leucine motif in determining the distribution of GIRK5 to the animal and vegetal poles. We found that Y16 and I22 contributed to functional expression and were dominant in the polarization of GIRK5. We thus conclude that the N-terminal acidic di-leucine motif of GIRK5 determines its retention and polarized trafficking within Xl oocytes.

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“…The open reading frames (ORF) of GIRK5 (U42207), Δ5GIRK5 and Δ25GIRK5 cDNAs were obtained by PCR as in previous studies [18,19]. Site‐specific mutagenesis of tyrosine 16 to an alanine residue (GIRK5Y16A and Δ5GIRK5Y16A) was introduced by the polymerase chain reaction using the mutant primers: (a) 5′ AAA GAT TGG CTG AGT CAC C 3′ (sense) and (b) 5′ GGT GAC TCA GCC AAT CTT T 3′ (antisense).…”

Section: Methodsmentioning

confidence: 99%

“…1 A). Δ25GIRK5 gives rise to functional channels with an outstanding basal activity (non‐receptor coupled) in oocytes [18] and insect cells [19]. Intriguingly, the isoforms GIRK5 and Δ5GIRK5 are not functional.…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation of a tyrosine at the N‐terminus regulates the surface expression of GIRK5 homomultimers

Mora

Escobar

2005

FEBS Letters

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The G protein-coupled inwardly rectifying GIRK5 and D5GIRK5 splicing variants do not express functional potassium channels. In contrast, D25GIRK5 forms functional homomultimers in Xenopus laevis oocytes. A tyrosine is present at the N-term of the non-functional isoforms. We studied the effect of endogenous tyrosine phosphorylation on the GIRK5 surface and functional expression. Unlike wild type channels, GIR-K5Y16A and D5GIRK5Y16A mutants displayed inwardly rectifying currents and inhibitors of Src tyrosine kinase promoted the traffiking of GIRK5 to the cell surface. This is the first evidence that endogenous phosphorylation of a tyrosine residue in a GIRK channel inhibits its surface expression.

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Functional properties of a truncated recombinant GIRK5 potassium channel

Cited by 9 publications

References 23 publications

Splitting the Two Pore Domains from TOK1 Results in Two Cationic Channels with Novel Functional Properties

Splitting the Two Pore Domains from TOK1 Results in Two Cationic Channels with Novel Functional Properties

The Polarization of the G-Protein Activated Potassium Channel GIRK5 to the Vegetal Pole of Xenopus laevis Oocytes Is Driven by a Di-Leucine Motif

Phosphorylation of a tyrosine at the N‐terminus regulates the surface expression of GIRK5 homomultimers

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