Limits...
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

Basal and agonist-evoked GIRK currents in neurons and oocytes are inversely related.(A) A representative whole-recording of GIRK current in a neuron. Switching from low-K+ extracellular solution to a high-K+ solution led to the development of a large inward current probably carried by several ion channel types. Addition of baclofen elicited Ievoked. Arrows show the amplitudes of Ibasal, Ievoked and Itotal. Extent of activation, Ra, is defined as Itotal/Ibasal. (B) Inverse correlation between Ibasal and Ra in oocytes and neurons. To allow direct comparison of Ibasal in oocytes and neurons, currents in neurons were corrected for the 10 mV difference in holding potential, which was -70 mV in neurons and -80 mV in oocytes (see Methods). The correlation between Ra and Ibasal was highly significant, p = 0.000000028 (neurons; n = 60; correlation coefficient = -0.633) and p = 0.0000002 (oocytes; n = 272; correlation coefficient = -0.728) by Spearman correlation test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4636287&req=5

pcbi.1004598.g001: Basal and agonist-evoked GIRK currents in neurons and oocytes are inversely related.(A) A representative whole-recording of GIRK current in a neuron. Switching from low-K+ extracellular solution to a high-K+ solution led to the development of a large inward current probably carried by several ion channel types. Addition of baclofen elicited Ievoked. Arrows show the amplitudes of Ibasal, Ievoked and Itotal. Extent of activation, Ra, is defined as Itotal/Ibasal. (B) Inverse correlation between Ibasal and Ra in oocytes and neurons. To allow direct comparison of Ibasal in oocytes and neurons, currents in neurons were corrected for the 10 mV difference in holding potential, which was -70 mV in neurons and -80 mV in oocytes (see Methods). The correlation between Ra and Ibasal was highly significant, p = 0.000000028 (neurons; n = 60; correlation coefficient = -0.633) and p = 0.0000002 (oocytes; n = 272; correlation coefficient = -0.728) by Spearman correlation test.

Mentions: First, we wanted to characterize the relation between GIRK’s Ibasal and Ievoked in hippocampal neurons, known to preferentially express GIRK1/2 [2]. Ibasal and Ievoked were measured using standard experimental paradigms ([39,47]; Fig 1A). Baclofen was used to activate the endogenous GABAB receptor and to generate Ievoked [52]. Net GIRK’s Ibasal was revealed as shown in Fig 1A (see also S1 Fig), by adding 100–120 nM tertiapin-Q (TPNQ), which selectively blocks >90% of GIRK currents in hippocampal neurons [39,43,53].


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)

Basal and agonist-evoked GIRK currents in neurons and oocytes are inversely related.(A) A representative whole-recording of GIRK current in a neuron. Switching from low-K+ extracellular solution to a high-K+ solution led to the development of a large inward current probably carried by several ion channel types. Addition of baclofen elicited Ievoked. Arrows show the amplitudes of Ibasal, Ievoked and Itotal. Extent of activation, Ra, is defined as Itotal/Ibasal. (B) Inverse correlation between Ibasal and Ra in oocytes and neurons. To allow direct comparison of Ibasal in oocytes and neurons, currents in neurons were corrected for the 10 mV difference in holding potential, which was -70 mV in neurons and -80 mV in oocytes (see Methods). The correlation between Ra and Ibasal was highly significant, p = 0.000000028 (neurons; n = 60; correlation coefficient = -0.633) and p = 0.0000002 (oocytes; n = 272; correlation coefficient = -0.728) by Spearman correlation test.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004598.g001: Basal and agonist-evoked GIRK currents in neurons and oocytes are inversely related.(A) A representative whole-recording of GIRK current in a neuron. Switching from low-K+ extracellular solution to a high-K+ solution led to the development of a large inward current probably carried by several ion channel types. Addition of baclofen elicited Ievoked. Arrows show the amplitudes of Ibasal, Ievoked and Itotal. Extent of activation, Ra, is defined as Itotal/Ibasal. (B) Inverse correlation between Ibasal and Ra in oocytes and neurons. To allow direct comparison of Ibasal in oocytes and neurons, currents in neurons were corrected for the 10 mV difference in holding potential, which was -70 mV in neurons and -80 mV in oocytes (see Methods). The correlation between Ra and Ibasal was highly significant, p = 0.000000028 (neurons; n = 60; correlation coefficient = -0.633) and p = 0.0000002 (oocytes; n = 272; correlation coefficient = -0.728) by Spearman correlation test.
Mentions: First, we wanted to characterize the relation between GIRK’s Ibasal and Ievoked in hippocampal neurons, known to preferentially express GIRK1/2 [2]. Ibasal and Ievoked were measured using standard experimental paradigms ([39,47]; Fig 1A). Baclofen was used to activate the endogenous GABAB receptor and to generate Ievoked [52]. Net GIRK’s Ibasal was revealed as shown in Fig 1A (see also S1 Fig), by adding 100–120 nM tertiapin-Q (TPNQ), which selectively blocks >90% of GIRK currents in hippocampal neurons [39,43,53].

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