Critical Determinants of the G Protein γ Subunits in the Gβγ Stimulation of G Protein-activated Inwardly Rectifying Potassium (GIRK) Channel Activity

Potassium channel activity of the basolateral membrane of the collagenase-treated rabbit proximal convoluted tubule (PCT) was studied during continuous luminal microperfusion. In cell-attached patches (high-K pipette) an inwardly rectifying potassium channel was observed with an inward slope conductance of 60.8 +/- 3.3 pS (n = 12) and outward slope conductance of 17.1 +/- 2.7 pS (n = 6). Stimulation of transcellular sodium transport with luminal glucose and alanine increased channel activity [measured as single-channel open probability (NPo)] from 0.19 +/- 0.11 to 0.44 +/- 0.09 (n = 8). This increase in channel activity was not likely to be mediated by either cell depolarization or cell swelling, because channel activity was voltage insensitive over physiological potentials and because the channel was not activated by stretch. However, channel activity was pH sensitive; reducing luminal pH from 7.4 to 6.5 reduced NPo from 0.63 +/- 0.24 to 0.26 +/- 0.16 (n = 5). Our work demonstrates the feasibility of patch clamping the basolateral membrane of microperfused nephron segments. This has allowed us to follow the activity of this potassium channel during an increase in sodium transport and show that its activity does increase during this maneuver. We conclude that: 1) it is possible to patch clamp the basolateral membrane of microperfused nephron segments, and 2) basolateral membrane of the rabbit PCT contains an inwardly rectifying, pH-sensitive potassium channel. The behavior of this channel on stimulation of transcellular sodium transport could explain the macroscopic increase in basolateral potassium conductance observed under similar conditions.

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Identification of Critical Residues Controlling G Protein-gated Inwardly Rectifying K+Channel Activity through Interactions with the βγ Subunits of G Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m104851200 ◽

2001 ◽

Vol 277 (8) ◽

pp. 6088-6096 ◽

Cited By ~ 79

Author(s):

Cheng He ◽

Xixin Yan ◽

Hailin Zhang ◽

Tooraj Mirshahi ◽

Taihao Jin ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

K Channel ◽

Channel Activity ◽

Critical Residues ◽

Inwardly Rectifying

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Rapid desensitization of G protein-gated inwardly rectifying K+ currents is determined by G protein cycle

AJP Cell Physiology ◽

10.1152/ajpcell.00540.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. C182-C191 ◽

Cited By ~ 21

Author(s):

Joanne L. Leaney ◽

Amy Benians ◽

Sean Brown ◽

Muriel Nobles ◽

David Kelly ◽

...

Keyword(s):

G Protein ◽

Competitive Inhibition ◽

Similar Degree ◽

Channel Activation ◽

Girk Channels ◽

Membrane Hyperpolarization ◽

Hek 293 ◽

Fast Activation ◽

Inwardly Rectifying ◽

Stimulation Of

Activation of G protein-gated inwardly rectifying K+ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of Gi/o-coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several Gi/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A1, adrenergic α2A, dopamine D2S, M4 muscarinic, and GABAB1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A1 receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5′- O-(3-thiotriphosphate) (GTPγS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca2+ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/GTP exchange by using nonhydrolyzable guanosine 5′- O-(2-thiodiphosphate) (GDPβS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific Gα subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle.

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Human Adrenal Glomerulosa Cells Express K2P and GIRK Potassium Channels that are inhibited by AngII and ACTH

AJP Cell Physiology ◽

10.1152/ajpcell.00118.2021 ◽

2021 ◽

Author(s):

John J. Enyeart ◽

Judith A. Enyeart

Keyword(s):

G Protein ◽

Zona Glomerulosa ◽

Time Dependent ◽

Aldosterone Secretion ◽

Girk Channels ◽

Whole Cell ◽

Whole Cell Patch Clamp ◽

Girk Channel ◽

Inwardly Rectifying ◽

G Protein Coupled

In whole-cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two pore (K2P) leak-type and a G-protein-coupled, inwardly-rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly-rectifying current was activated by arachidonic acid and inhibited by angiotensin II (AngII), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by AngII, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by AngII or ACTH, and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by AngII and ACTH are likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

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G-protein–gated inwardly rectifying potassium channels regulate ADP-induced cPLA2 activity in platelets through Src family kinases

Blood ◽

10.1182/blood-2006-03-010330 ◽

2006 ◽

Vol 108 (9) ◽

pp. 3027-3034 ◽

Cited By ~ 23

Author(s):

Haripriya Shankar ◽

Bryan N. Kahner ◽

Janani Prabhakar ◽

Parth Lakhani ◽

Soochong Kim ◽

...

Keyword(s):

Potassium Channels ◽

G Protein ◽

P2y Receptors ◽

Null Mouse ◽

Channel Blockers ◽

Functional Responses ◽

Kinase Activation ◽

Girk Channel ◽

Inwardly Rectifying Potassium Channels ◽

Inwardly Rectifying

Abstract ADP-induced TXA2 generation requires the costimulation of P2Y1, P2Y12, and the GPIIb/IIIa receptors. Signaling events downstream of the P2Y receptors that contribute to ADP-induced TXA2 generation have not been clearly delineated. In this study, we have investigated the role of G-protein–gated inwardly rectifying potassium channels (GIRKs), a recently identified functional effector for the P2Y12 receptor, in the regulation of ADP-induced TXA2 generation. At 10-μM concentrations, the 2 structurally distinct GIRK channel blockers, SCH23390 and U50488H, caused complete inhibition of ADP-induced cPLA2 phosphorylation and TXA2 generation, without affecting the conversion of AA to TXA2 or ADP-induced primary platelet aggregation in aspirin-treated platelets. In addition, Src family kinase selective inhibitors abolished 2MeSADP-mediated cPLA2 phosphorylation and TXA2 generation. Furthermore, these GIRK channel blockers completely blocked Gi-mediated Src kinase activation, suggesting that GIRK channels are upstream of Src family tyrosine kinase activation. In weaver mouse platelets, which have dysfunctional GIRK2 subunits, ADP-induced TXA2 generation was impaired. However, we did not observe any defect in 2MeSADP-induced platelet functional responses in GIRK2-null mouse platelets, suggesting that functional channels composed of other GIRK subunits contribute to ADP-induced TXA2 generation, via the regulation of the Src and cPLA2 activity.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.1.h379 ◽

1996 ◽

Vol 271 (1) ◽

pp. H379-H385 ◽

Cited By ~ 3

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.

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