Functional Characterization of a Prokaryotic Kir Channel

Enkvetchakul, Decha; Bhattacharyya, Jaya; Jeliazkova, Iana; Groesbeck, Darcy K.; Cukras, Catherine A; Nichols, Colin G.

doi:10.1074/jbc.c400417200

Cited by 54 publications

(76 citation statements)

References 24 publications

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“…Methods are essentially as described previously (22). KcsA and KirBac1.1 in pQE60 vector were expressed in BL21* (DE3) cells after induction with isopropyl ␤-D-thiogalactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

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Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Enkvetchakul

Jeliazkova

Nichols

2005

Journal of Biological Chemistry

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Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86 Rb ؉ uptake assay. We demonstrate direct modulation by PIP 2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP 2 , KirBac1.1 is inhibited by PIP 2 incorporated in the membrane (K1 ⁄ 2 ‫؍‬ 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP 2 in the membrane.Phosphoinosotides constitute a major group of signaling molecules in eukaryotic membranes (1, 2) and modulate an ever growing list of ion channels, whether by application of exogenous phospholipids to the cytoplasmic membrane surface or by manipulation of endogenous phospholipids (3-12). However, the nature of the phosphoinositide-channel interaction remains elusive. For one extensively studied group, the inward rectifier K (Kir) channels, there is an emerging consensus that a direct interaction occurs between cytoplasmic domains of the channel and inositol headgroups, based on electrophysiological analysis (5, 13-16) and biochemical analysis of isolated channel domains (5,17,18). Direct interaction of functional channels with phospholipids in the membrane has been difficult to demonstrate unequivocally, and without this, quantification of the dose-response relationships for channel modulation by phospholipids is obviated, and further mechanistic understanding is limited (19, 20).The recent cloning and crystallization (21), as well as functional analysis of KirBac1.1 channels reconstituted in lipid membranes (22), provides the opportunity to examine channel activity using a highly purified protein preparation in membranes of defined composition and permits direct test of the nature of the channel-phosphoinositide interaction. EXPERIMENTAL PROCEDURESMethods are essentially as described previously (22). KcsA and KirBac1.1 in pQE60 vector were expressed in BL21* (DE3) cells after induction with isopropyl ␤-D-thiogalactopyranoside. Bacteria were lysed by sonication, incubated 2-4 h with 30 mM decylmaltoside (Anatrace), then centrifuged at 30,000 ϫ g for 30 min, and the supernatant was applied to a cobalt affinity column. The column was washed with 20 -30 volumes of wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM KCl, 10 mM imidazole, and 5 mM decylmaltoside) and eluted with 1-2 ml of wash buffer containing 500 mM imidazole. Protein was concentrated u...

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“…Methods are essentially as described previously (22). KcsA and KirBac1.1 in pQE60 vector were expressed in BL21* (DE3) cells after induction with isopropyl ␤-D-thiogalactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

“…The recent cloning and crystallization (21), as well as functional analysis of KirBac1.1 channels reconstituted in lipid membranes (22), provides the opportunity to examine channel activity using a highly purified protein preparation in membranes of defined composition and permits direct test of the nature of the channel-phosphoinositide interaction.…”

mentioning

confidence: 99%

Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Enkvetchakul

Jeliazkova

Nichols

2005

Journal of Biological Chemistry

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“…In contrast, recent X-ray crystal structures of the prokaryotic KirBac channels KirBac1.1 (12) and KirBac3.1 (18) have revealed that these channels share extensive structural homology with the superfamily of mammalian Kir channels and can be regarded as true "prokaryotic homologs" (2). However, despite this wealth of structural information, little functional data exist for prokaryotic KirBac channels; the only functional studies have been macroscopic ion flux studies using purified KirBac1.1 protein in reconstituted liposomes (4,5). Although these studies have been useful, they are labor intensive and are not particularly suited to high-throughput analysis.…”

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confidence: 99%

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

Sun

Gan

Paynter

et al. 2006

Physiological Genomics

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“…MmaK Is Inactivated by Slightly Acidic pH but Only from the Cytoplasmic Side-Several prokaryotic K ϩ channels are known to be regulated by pH (12,17,27,28). To see whether the activity of MmaK is also modulated by pH, we perfused the cytoplasmic-side-out membrane patch with perfusates containing various pH values from 8.5 to 5.5.…”

Section: Mmak Conducts An Inwardly Rectified Kmentioning

confidence: 99%

“…A common strategy has been reconstitution of the purified channel protein into artificial lipids for bilayer lipid membrane measurement (5,10,11), a process that relies on the chance survival of the channel during detergent extraction and lipid reconstitution. In some cases, the reconstituted channel activities can only be demonstrated with the low resolution 86 Rb ϩ uptake assay (9,(12)(13)(14). An often overlooked opportunity to capture these channels in action is the very membrane of the E. coli cells in which they are commonly overproduced.…”

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confidence: 99%

Patch Clamp and Phenotypic Analyses of a Prokaryotic Cyclic Nucleotide-gated K+ Channel Using Escherichia coli as a Host

Kuo

Saimi

Kung

et al. 2007

Journal of Biological Chemistry

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Prokaryotic ion channels have been valuable in providing structural models for understanding ion filtration and channel-gating mechanisms. However, their functional examinations have remained rare and usually been carried out by incorporating purified channel protein into artificial lipid membranes. Here we demonstrate the utilization of Escherichia coli to host the functional analyses by examining a putative cyclic nucleotide-gated K ؉ channel cloned from Magnetospirillum magnetotacticum, MmaK. When expressed in wild-type E. coli cells, MmaK renders the host sensitive to millimolar concentrations of externally applied K ؉ , indicating MmaK forms a functional K ؉ conduit in the E. coli membrane in vivo. After enlarging these cells into giant spheroplasts, macro-and microscopic MmaK currents are readily detected in excised E. coli membrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and ϳ10-fold higher concentration of cGMP and manifests as an inwardly rectified, K ؉ -specific current with a 10.8 pS unitary conductance at ؊100 mV. Additionally, MmaK is inactivated by slightly acidic pH only from the cytoplasmic side. Our in vitro biophysical characterizations of MmaK correlate with its in vivo phenotype in E. coli, implicating its critical role as an intracellular cAMP and pH sensor for modulating bacterial membrane potential. Exemplified by MmaK functional studies, we establish that E. coli and its giant spheroplast provide a convenient and versatile system to express foreign channels for biophysical analyses that can be further dovetailed with microbial genetics.Recent sequencing of bacterial genomes reveals that ion channels evolved as early as three billion years ago. K ϩ channels, for example, are widely spread in all life forms, Bacteria, Archaea, and Eukarya (1, 2). Because prokaryotic channel genes can often be heterologously expressed in Escherichia coli at high yield, the channel proteins so produced have laid an inroad to determine their crystal structures. Beginning with the prelude of MacKinnon and Doyle (3) crystal structures of these channels have raised our understanding of the molecular bases of ion channels as illustrated in several atomic structures of prokaryotic K ϩ channels (4 -9).Functional interpretation of prokaryotic channel structures by electrophysiological methods, however, has not been straightforward. The main technical barrier is that the prokaryotic channel activities are often difficult to analyze under the existing methodology. A common strategy has been reconstitution of the purified channel protein into artificial lipids for bilayer lipid membrane measurement (5, 10, 11), a process that relies on the chance survival of the channel during detergent extraction and lipid reconstitution. In some cases, the reconstituted channel activities can only be demonstrated with the low resolution 86 Rb ϩ uptake assay (9, 12-14). An often overlooked opportunity to capture these channels in action is the very membrane of the E. coli cells in which they a...

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Functional Characterization of a Prokaryotic Kir Channel

Cited by 54 publications

References 24 publications

Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

Patch Clamp and Phenotypic Analyses of a Prokaryotic Cyclic Nucleotide-gated K+ Channel Using Escherichia coli as a Host

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