scholarly journals Functional expression of a vertebrate inwardly rectifying K+ channel in yeast.

1995 ◽  
Vol 6 (9) ◽  
pp. 1231-1240 ◽  
Author(s):  
W Tang ◽  
A Ruknudin ◽  
W P Yang ◽  
S Y Shaw ◽  
A Knickerbocker ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Xenopus Oocytes ◽  
Genetic System ◽  
Functional Expression ◽  
Inward Rectifier ◽  
K Channel ◽  
Coding Region ◽  
Low Potassium ◽  
K Current ◽  
Inwardly Rectifying

We describe the expression of gpIRK1, an inwardly rectifying K+ channel obtained from guinea pig cardiac cDNA. gpIRK1 is a homologue of the mouse IRK1 channel identified in macrophage cells. Expression of gpIRK1 in Xenopus oocytes produces inwardly rectifying K+ current, similar to the cardiac inward rectifier current IK1. This current is blocked by external Ba2+ and Cs+. Plasmids containing the gpIRK1 coding region under the transcriptional control of constitutive (PGK) or inducible (GAL) promoters were constructed for expression in Saccharomyces cerevisiae. Several observations suggest that gpIRK1 forms functional ion channels when expressed in yeast. gpIRK1 complements a trk1 delta trk2 delta strain, which is defective in potassium uptake. Expression of gpIRK1 in this mutant restores growth on low potassium media. Growth dependent on gpIRK1 is inhibited by external Cs+. The strain expressing gpIRK1 provides a versatile genetic system for studying the assembly and composition of inwardly rectifying K+ channels.

Intracellular H+ inhibits a cloned rat kidney outer medulla K+ channel expressed in Xenopus oocytes

1995 ◽  
Vol 268 (5) ◽  
pp. C1173-C1178 ◽  
Author(s):  
T. D. Tsai ◽  
M. E. Shuck ◽  
D. P. Thompson ◽  
M. J. Bienkowski ◽  
K. S. Lee
Keyword(s):  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Ph Sensitivity ◽  
Resting Potential ◽  
K Channel ◽  
K Current ◽  
A Site ◽  
The Relationship ◽  
Inwardly Rectifying ◽  
External Ph

The pH sensitivity of a cloned rat kidney K+ channel, ROMK1, was examined after expression in Xenopus oocytes. Membrane currents and intracellular pH (pHi) were concomitantly monitored by the two-microelectrode voltage-clamp technique and a pH-sensitive microelectrode. Oocytes injected with ROMK1 cRNA developed a hyperpolarized resting potential of -98.7 +/- 0.98 mV and a slightly inwardly rectifying Ba(2+)-sensitive K+ current. Lowering external pH from 7.4 to 6.7 using membrane-permeable acetate buffer reduced measured pHi from 7.2 to 6.6 and reduced the ROMK1 current by 80%. The H+ blockade of ROMK1 currents was voltage independent. The relationship between ROMK1 slope conductance and pHi fitted to a titration curve suggested binding of four H+ to a site with a pK of 6.79. Extracellular acidification from pH 7.4 to 6.0 using membrane-impermeable biphthalate buffer had no effect on the ROMK1 current. The pH sensitivity of the ROMK1 channel is similar to that reported for a small-conductance native kidney K+ channel.

Muscarine-gated K+ channel: subunit stoichiometry and structural domains essential for G protein stimulation

1996 ◽  
Vol 271 (1) ◽  
pp. H379-H385 ◽  
Author(s):  
S. J. Tucker ◽  
M. Pessia ◽  
J. P. Adelman
Keyword(s):  
G Protein ◽  
Xenopus Oocytes ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Structural Domains ◽  
Maximal Stimulation ◽  
Subunit Stoichiometry ◽  
K Current ◽  
Inwardly Rectifying

Coexpression in Xenopus oocytes of the cloned cardiac inward rectifier subunits Kir 3.1 and Kir 3.4 results in G protein-stimulated channel activity closely resembling the muscarinic channel underlying the inwardly rectifying K+ current in atrial myocytes. To determine the stoichiometry and relative subunit positions within the channel, Kir 3.1 and Kir 3.4 were coexpressed in varying ratios with cloned G beta 1 gamma 2 subunits and also as tandemly linked tetramers with different relative subunit positions. The results reveal that the most efficient channel comprises two subunits of each type in an alternating array within the tetramer. To localize regions important for subunit coassembly and G protein sensitivity, chimeric subunits containing domains from either Kir 3.1, Kir 3.4, or the G protein-insensitive subunit Kir 4.1 were expressed. The results demonstrate that the transmembrane domains dictate the potentiation of the coassembled channels and that, although the NH4- or COOH-termini of both subunits alone can confer G protein sensitivity, both termini are required for maximal stimulation by G beta 1 gamma 2.

scholarly journals Primary structure and functional expression of a mouse inward rectifier K+ channel and rat G-protein-coupled muscarinic K+ channel.

1995 ◽  
Vol 15 (2) ◽  
pp. 106-113
Author(s):  
Yoshihiro Kubo ◽  
Eitan Reuveny ◽  
Paul A Slesinger ◽  
Timothy J Baldwin ◽  
Yuh Nung Jan ◽  
...  
Keyword(s):  
G Protein ◽  
Primary Structure ◽  
Functional Expression ◽  
Inward Rectifier ◽  
K Channel ◽  
G Protein Coupled

scholarly journals Cloning and functional expression of a novel iso-form of ROMK inwardly rectifying ATP-dependent K+ channel. ROMK6 (KIrl.1f)

1997 ◽  
Vol 73 ◽  
pp. 81
Author(s):  
Chikako Kondo ◽  
Shojiro Isomoto ◽  
Shigeto Matsumoto ◽  
Mitstihiko Yamada ◽  
Yoshiyuki Horio ◽  
...  
Keyword(s):  
Functional Expression ◽  
K Channel ◽  
Inwardly Rectifying

ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and distribution of alternative forms

1995 ◽  
Vol 268 (6) ◽  
pp. F1132-F1140 ◽  
Author(s):  
M. A. Boim ◽  
K. Ho ◽  
M. E. Shuck ◽  
M. J. Bienkowski ◽  
J. H. Block ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Functional Expression ◽  
Amino Acid Sequences ◽  
K Channel ◽  
K Channels ◽  
Noncoding Regions ◽  
Nephron Segments ◽  
Alternatively Spliced ◽  
Polymerase Chain ◽  
Inwardly Rectifying

The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channels [K. Ho, C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Nature Lond. 362: 31–38, 1993; H. Zhou, S. S. Tate, and L. G. Palmer. Am. J. Physiol. 266 (Cell Physiol. 35): C809-C824, 1994], and mRNA encoding these channels is widely expressed in distal cortical and outer medullary nephron segments [see companion study; W.-S. Lee and S. C. Hebert. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1124-F1131, 1995]. Using approaches based on homology to ROMK1, we have identified two additional ROMK isoforms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is due to alternative splicing at both the 5'-coding and 3'-noncoding regions. The splicing at the 5' end of ROMK gives rise to channel proteins with variable-length NH2 termini containing different initial amino acid sequences. Functional expression of these isoforms in Xenopus oocytes showed that they form functional Ba(2+)-sensitive K+ channels. The nephron distribution of mRNAs encoding alternatively spliced isoforms of ROMK (ROMK1-ROMK3) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of nephron segments dissected from rat kidney. Nondegenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cloning and functional expression of a novel G-protein-gated inwardly rectifying K+ channel subunit, G1RK2B.

1996 ◽  
Vol 71 ◽  
pp. 150
Author(s):  
Shojiro Isomoto ◽  
Chikako Kondo ◽  
Naohiko Takahashi ◽  
Shigeto Matsumoto ◽  
Mitsuhiko Yamada ◽  
...  
Keyword(s):  
G Protein ◽  
Functional Expression ◽  
K Channel ◽  
Inwardly Rectifying

scholarly journals A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes.

1995 ◽  
Vol 105 (3) ◽  
pp. 421-439 ◽  
Author(s):  
N F Lim ◽  
N Dascal ◽  
C Labarca ◽  
N Davidson ◽  
H A Lester
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Xenopus Oocytes ◽  
K Channel ◽  
Intracellular Camp ◽  
Heterotrimeric G Proteins ◽  
M2 Muscarinic Receptor ◽  
Gamma Subunits ◽  
Beta 2 ◽  
Inwardly Rectifying

In many tissues, inwardly rectifying K channels are coupled to seven-helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.

scholarly journals Functional expression of a Ca2+-activated K+ channel in Xenopus oocytes injected with RNAs from the rat testis

1998 ◽  
Vol 1373 (2) ◽  
pp. 360-365 ◽  
Author(s):  
W.L. Wu ◽  
S.C. So ◽  
Y.P. Sun ◽  
T.S. Zhou ◽  
Y. Yu ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Functional Expression ◽  
K Channel ◽  
Rat Testis
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