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

Yakubovich D, Berlin S, Kahanovitch U, Rubinstein M, Farhy-Tselnicker I, Styr B, Keren-Raifman T, Dessauer CW, Dascal N - PLoS Comput. Biol. (2015)

Bottom Line: Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter.In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
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 GIRK1/2's 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.

No MeSH data available.


Related in: MedlinePlus

Gating of GIRK1/2 by Gβγ.(A) Sources of Gβγ for GIRK activation. GαGDP●Gβγ is the undissociated G protein heterotrimer. Note that, in isolated Xenopus oocytes or HEK cells, in the absence of added agonist the right, GPCR-dependent branch of the reaction of Fig 2A does not significantly contribute to Ibasal, because there are no known Gαi/o-coupled GPCRs or ambient agonists that can "basally" activate the GTPase cycle (discussed in [51]). (B) The schemes of “concerted”, “graded contribution” and “separate gating transitions” models of channel activation. (C) Graded contribution of the four Gβγ-occupied GIRK states to Po. Fractional Po for each state was calculated by normalizing published Po values [13] of each of the four modes (corresponding to 1–4 Gβγ occupied state) to Po,max (corresponding to 4 Gβγ occupied channel). Almost identical values have been obtained from fractional activation ratios for engineered GIRK channels having 1 to 4 Gβγ binding sites [14].
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pcbi.1004598.g002: Gating of GIRK1/2 by Gβγ.(A) Sources of Gβγ for GIRK activation. GαGDP●Gβγ is the undissociated G protein heterotrimer. Note that, in isolated Xenopus oocytes or HEK cells, in the absence of added agonist the right, GPCR-dependent branch of the reaction of Fig 2A does not significantly contribute to Ibasal, because there are no known Gαi/o-coupled GPCRs or ambient agonists that can "basally" activate the GTPase cycle (discussed in [51]). (B) The schemes of “concerted”, “graded contribution” and “separate gating transitions” models of channel activation. (C) Graded contribution of the four Gβγ-occupied GIRK states to Po. Fractional Po for each state was calculated by normalizing published Po values [13] of each of the four modes (corresponding to 1–4 Gβγ occupied state) to Po,max (corresponding to 4 Gβγ occupied channel). Almost identical values have been obtained from fractional activation ratios for engineered GIRK channels having 1 to 4 Gβγ binding sites [14].

Mentions: We start the development of the model by considering how Gβγ, available for activation of GIRK, can be derived from heterotrimeric Gαβγ (Fig 2A). In the absence of GPCR-activated G protein cycle, a small fraction of G proteins dissociates into free GαGDP and Gβγ due to finite affinity of their interaction [55,56] (the left branch of the reaction in Fig 2A). This free Gβγ can contribute to Ibasal [57,58]. Addition of agonist activates the GPCR and promotes GDP-GTP exchange at Gα and full or partial separation of GαGTP from Gβγ (the right branch of the reaction in Fig 2A; [59,60,61]). In our experiments in Xenopus oocytes and HEK293 cells, we coexpressed the muscarinic receptor 2 (m2R) which couples to Gi/o, and used acetylcholine (ACh) at supramaximal doses [62], 2–10 μM, in order to achieve a complete separation/rearrangement between GαGTP and Gβγ [63,64].


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

Yakubovich D, Berlin S, Kahanovitch U, Rubinstein M, Farhy-Tselnicker I, Styr B, Keren-Raifman T, Dessauer CW, Dascal N - PLoS Comput. Biol. (2015)

Gating of GIRK1/2 by Gβγ.(A) Sources of Gβγ for GIRK activation. GαGDP●Gβγ is the undissociated G protein heterotrimer. Note that, in isolated Xenopus oocytes or HEK cells, in the absence of added agonist the right, GPCR-dependent branch of the reaction of Fig 2A does not significantly contribute to Ibasal, because there are no known Gαi/o-coupled GPCRs or ambient agonists that can "basally" activate the GTPase cycle (discussed in [51]). (B) The schemes of “concerted”, “graded contribution” and “separate gating transitions” models of channel activation. (C) Graded contribution of the four Gβγ-occupied GIRK states to Po. Fractional Po for each state was calculated by normalizing published Po values [13] of each of the four modes (corresponding to 1–4 Gβγ occupied state) to Po,max (corresponding to 4 Gβγ occupied channel). Almost identical values have been obtained from fractional activation ratios for engineered GIRK channels having 1 to 4 Gβγ binding sites [14].
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636287&req=5

pcbi.1004598.g002: Gating of GIRK1/2 by Gβγ.(A) Sources of Gβγ for GIRK activation. GαGDP●Gβγ is the undissociated G protein heterotrimer. Note that, in isolated Xenopus oocytes or HEK cells, in the absence of added agonist the right, GPCR-dependent branch of the reaction of Fig 2A does not significantly contribute to Ibasal, because there are no known Gαi/o-coupled GPCRs or ambient agonists that can "basally" activate the GTPase cycle (discussed in [51]). (B) The schemes of “concerted”, “graded contribution” and “separate gating transitions” models of channel activation. (C) Graded contribution of the four Gβγ-occupied GIRK states to Po. Fractional Po for each state was calculated by normalizing published Po values [13] of each of the four modes (corresponding to 1–4 Gβγ occupied state) to Po,max (corresponding to 4 Gβγ occupied channel). Almost identical values have been obtained from fractional activation ratios for engineered GIRK channels having 1 to 4 Gβγ binding sites [14].
Mentions: We start the development of the model by considering how Gβγ, available for activation of GIRK, can be derived from heterotrimeric Gαβγ (Fig 2A). In the absence of GPCR-activated G protein cycle, a small fraction of G proteins dissociates into free GαGDP and Gβγ due to finite affinity of their interaction [55,56] (the left branch of the reaction in Fig 2A). This free Gβγ can contribute to Ibasal [57,58]. Addition of agonist activates the GPCR and promotes GDP-GTP exchange at Gα and full or partial separation of GαGTP from Gβγ (the right branch of the reaction in Fig 2A; [59,60,61]). In our experiments in Xenopus oocytes and HEK293 cells, we coexpressed the muscarinic receptor 2 (m2R) which couples to Gi/o, and used acetylcholine (ACh) at supramaximal doses [62], 2–10 μM, in order to achieve a complete separation/rearrangement between GαGTP and Gβγ [63,64].

Bottom Line: Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter.In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
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 GIRK1/2's 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.

No MeSH data available.


Related in: MedlinePlus