scholarly journals A unique P-region residue is required for slow voltage-dependent gating of a G protein-activated inward rectifier K+ channel expressed in Xenopus oocytes.

1996 ◽  
Vol 490 (3) ◽  
pp. 633-645 ◽  
Author(s):  
P Kofuji ◽  
C A Doupnik ◽  
N Davidson ◽  
H A Lester
Keyword(s):  
G Protein ◽  
Xenopus Oocytes ◽  
Inward Rectifier ◽  
K Channel ◽  
Voltage Dependent

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.

scholarly journals Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 430
Author(s):  
Evan Van Aalst ◽  
Maryam Yekefallah ◽  
Anil K. Mehta ◽  
Isaac Eason ◽  
Benjamin Wylie
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Recombinant Expression ◽  
Protein Product ◽  
Inward Rectifier ◽  
Correlation Spectroscopy ◽  
Protein Yield ◽  
K Channel ◽  
Functional Assay ◽  
Nmr Studies

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

scholarly journals Subunit Stoichiometry of a Heteromultimeric G protein-coupled Inward-rectifier K+Channel

1996 ◽  
Vol 271 (48) ◽  
pp. 30524-30528 ◽  
Author(s):  
Scott K. Silverman ◽  
Henry A. Lester ◽  
Dennis A. Dougherty
Keyword(s):  
G Protein ◽  
Inward Rectifier ◽  
K Channel ◽  
Subunit Stoichiometry ◽  
G Protein Coupled

scholarly journals Intrinsic gating properties of a cloned G protein-activated inward rectifier K+ channel.

1995 ◽  
Vol 106 (1) ◽  
pp. 1-23 ◽  
Author(s):  
C A Doupnik ◽  
N F Lim ◽  
P Kofuji ◽  
N Davidson ◽  
H A Lester
Keyword(s):  
G Protein ◽  
K Channel ◽  
Extrinsic Factors ◽  
Time Resolved ◽  
Time Constants ◽  
Protein Activation ◽  
G Protein Activation ◽  
Voltage Dependent ◽  
Current Activation ◽  
Inwardly Rectifying

The voltage-, time-, and K(+)-dependent properties of a G protein-activated inwardly rectifying K+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K+ (VK = 0 mV), voltage jumps from VK to negative membrane potentials activated inward GIRK1 K+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants tau f approximately 50 ms and tau s approximately 400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from VK. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to VK and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK -20 mV; the equivalent gating charge was approximately 1.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein beta 1 gamma 2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K+ channels.

scholarly journals Positive and Negative Coupling of the Metabotropic Glutamate Receptors to a G Protein–activated K+ Channel, GIRK, in Xenopus Oocytes

1997 ◽  
Vol 109 (4) ◽  
pp. 477-490 ◽  
Author(s):  
Dahlia Sharon ◽  
Dmitry Vorobiov ◽  
Nathan Dascal
Keyword(s):  
Protein Kinase ◽  
Glutamate Receptors ◽  
G Protein ◽  
G Proteins ◽  
Metabotropic Glutamate Receptors ◽  
Xenopus Oocytes ◽  
K Channel ◽  
Metabotropic Glutamate ◽  
Girk Channel ◽  
The Brain

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K+ channel, GIRK, is activated by the βγ subunits of Gi proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by Gi/Go-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the Gq class, inhibited the channel's activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca2+ chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-μ. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a Gi/Go protein. The observed modulations may be involved in the mGluRs' effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C–activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to Gq are inefficient in activating GIRK.

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 Mechanism of the Voltage Sensitivity of IRK1 Inward-rectifier K+ Channel Block by the Polyamine Spermine

2005 ◽  
Vol 125 (4) ◽  
pp. 413-426 ◽  
Author(s):  
Hyeon-Gyu Shin ◽  
Zhe Lu
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Head Groups ◽  
Voltage Dependent

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.

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