Daniel Yakubovich

Na+ Promotes the Dissociation between GαGDP and Gβγ, Activating G Protein-gated K+ Channels

G protein-gated K+channels (GIRK, or Kir3) are activated by the direct binding of Gβγ or of cytosolic Na+. Na+ activation is fast, Gβγ-independent, and probably via a direct, low affinity (EC50, 30–40 mm) binding of Na+ to the channel. Here we demonstrate that an increase in intracellular Na+ concentration, [Na+]in, within the physiological range (5–20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na+ effect. It is inhibited by a Gβγ scavenger, hence it is Gβγ-dependent; but it does not require GTP. We hypothesized that Na+ elevates the cellular concentration of free Gβγ by promoting the dissociation of the Gαβγ heterotrimer into free GαGDP and Gβγ. Direct biochemical measurements showed that Na+ causes a moderate decrease (∼2-fold) in the affinity of interaction between GαGDP and Gβγ. Furthermore, in accord with the predictions of our model, slow Na+ activation was enhanced by mild coexpression of Gαi3. Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gβγ-dependent regulation of GIRK by Na+. We propose that Na+ may act as a regulatory factor, or even a second messenger, that regulates effectors via Gβγ.

Gβγ-dependent and Gβγ-independent Basal Activity of G Protein-activated K+ Channels

Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gβγ subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gβγ, but a Gβγ-independent component has also been envisaged. We investigated Gβγ dependence of the basal GIRK activity (AGIRK,basal) quantitatively, by titrated expression of Gβγ scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gβγ scavenger, myristoylated C terminus of β-adrenergic kinase (m-cβARK), reduced AGIRK,basal by 70–80% and eliminated the acetylcholine-evoked current (IACh). However, we found that m-cβARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gβγ scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of AGIRK,basal by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gβγ and Gα in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gβγ and its constitutive association with GIRK may underlie the excessively high AGIRK,basal observed at high expression levels of GIRK. Only 10–15% of AGIRK,basal persisted upon expression of both m-phosducin and cβARK. These results demonstrate that a major part of Ibasal is Gβγ-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gβγ-independent.

Slow modal gating of single G protein‐activated K+ channels expressed in Xenopus oocytes

1 The slow kinetics of G protein‐activated K+ (GIRK) channels expressed in Xenopus oocytes were studied in single‐channel, inside‐out membrane patches. Channels formed by GIRK1 plus GIRK4 subunits, which are known to form the cardiac acetylcholine (ACh)‐activated GIRK channel (KACh), were activated by a near‐saturating dose of G protein βγ subunits (Gβγ; 20 nM). 2 The kinetic parameters of the expressed GIRK1/4 channels were similar to those of cardiac KACh. GIRK1/4 channels differed significantly from channels formed by GIRK1 with the endogenous oocyte subunit GIRK5 (GIRK1/5) in some of their kinetic parameters and in a 3‐fold lower open probability, Po. The unexpectedly low Po (0.025) of GIRK1/4 was due to the presence of closures of hundreds of milliseconds; the channel spent ∼90 % of the time in the long closed states. 3 GIRK1∼4 channels displayed a clear modal behaviour: on a time scale of tens of seconds, the Gβγ‐activated channels cycled between a low‐Po mode (Po of about 0.0034) and a bursting mode characterized by an ∼30‐fold higher Po and a different set of kinetic constants (and, therefore, a different set of channel conformations). The available evidence indicates that the slow modal transitions are not driven by binding and unbinding of Gβγ. 4 The GTPγS‐activated Gαi1 subunit, previously shown to inhibit GIRK channels, substantially increased the time spent in closed states and apparently shifted the channel to a mode similar, but not identical, to the low‐Po mode. 5 This is the first demonstration of slow modal transitions in GIRK channels. The detailed description of the slow gating kinetics of GIRK1∼4 may help in future analysis of mechanisms of GIRK gating.

Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein beta/gamma-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP(2)A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: (i) The open probability (P(o)) of GIRK1/GIRK4 channels, stimulated by coexpressed m(2)-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. (ii) At moderate concentrations of recombinant G(beta1/gamma2), used to activate the channel, P(o) was significantly reduced in patches treated with PP(2)A, when compared to patches with PKA-cs. (iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P(o) at moderate G-protein concentrations. (iv) An increase of the frequency of openings (f(o)) and a reduction of dwell time duration of the channel in the long-lasting C(5) state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP(2)A led to an increase of G(beta1/gamma2) concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G(beta1/gamma2). (v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP(2)A, indicating that they constitute an essential part of the off-switch.

Kinetic modeling of Na+-induced, Gβγ-dependent activation of G protein-gated K+ channels

G protein-activated K+(GIRK) channels are activated by numerous neurotransmitters that act on Gi/o proteins, via a direct interaction with the Gβγ subunit of G proteins. In addition, GIRK channels are positively regulated by intracellular Na+ via a direct interaction (fast pathway) and via a Gβγ-dependent mechanism (slow pathway). The slow modulation has been proposed to arise from the recently described phenomenon of Na+-induced reduction of affinity of interaction between GαGDP and Gβγ subunits of G proteins. In this scenario, elevated Na+ enhances basal dissociation of G protein heterotrimers, elevating free cellular Gβγ and activating GIRK. However, it is not clear whether this hypothesis can account for the quantitative and kinetic aspects of the observed regulation. Here, we report the development of a quantitative model of slow, Na+-dependent, G protein-mediated activation of GIRK. Activity of GIRK1F137S channels, which are devoid of direct interaction with Na+, was measured in excised membrane patches and used as an indicator of free Gβγ levels. The change in channel activity was used to calculate the Na+-dependent change in the affinity of G protein subunit interaction. Under a wide range of initial conditions, the model predicted that a relatively small decrease in the affinity of interaction of GαGDP and Gβγ (about twofold under most conditions) accounts for the twofold activation of GIRK induced by Na+, in agreement with biochemical data published previously. The model also correctly described the slow time course of Na+ effect and explained the previously observed enhancement of Na+-induced activation of GIRK by coexpressed Gαi3. This is the first quantitative model that describes the basal equilibrium between free and bound G protein subunits and its consequences on regulation of a Gβγ effector.

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ.

G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2s density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.

Amplitude Histogram-Based Method of Analysis of Patch Clamp Recordings that Involve Extreme Changes in Channel Activity Levels

Many ion channels show low basal activity, which is increased hundreds-fold by the relevant gating factor. A classical example is the activation G-protein-activated K+ channels (GIRK) by Gβγ subunit dimer. The extent of activation (relative to basal current), Ra, is an important physiological parameter, usually readily estimated from whole cell recordings. However, calculation of Ra often becomes non-trivial in multi-channel patches because of extreme changes in activity upon activation, from a seemingly single-channel pattern to a macroscopic one. In such cases, calculation of the net current flowing through the channels in the patch,

The Human Muscarinic Receptor Couples to GαI3 Via Catalytic Collision

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