Amy Benians | Otago Polytechnic (original) (raw)
Papers by Amy Benians
Scope: Contemporary Research Topics (Learning and Teaching), 2021
Proceedings of the National Academy of Sciences of the United States of America, Apr 28, 2003
G protein-gated inwardly rectifying K ؉ (Kir) channels are found in neurones, atrial myocytes, an... more G protein-gated inwardly rectifying K ؉ (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G proteincoupled receptors (GPCRs) via the inhibitory family of G protein, G i/o, in a membrane-delimited fashion by the direct binding of G␥ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K ؉ channel, Kir3.1 ؉ 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein ␣ subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein ␣ subunit. With some combinations of agonist͞GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein ␣ subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs. G protein ␣ subunit ͉ inward rectifier ͉ cell signaling ͉ drug development ͉ G protein-signaling protein
Journal of Biological Chemistry, Aug 1, 2002
Signaling studies in living cells would be greatly facilitated by the development of functional f... more Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein ␣ subunits. We have designed G i/o ␣ subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-proteingated inwardly rectifying K ؉ channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G i/o ␣ subunits (G i1 ␣, G i2 ␣, G i3 ␣, and G oA ␣) and a mutation introduced at Cys ؊4 rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G i/o ␣ protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G i/o ␣ subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the G␥ dimer by the mutant G␣ subunit. Moreover, each subunit was able to support A1mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G i3 ␣ and G oA ␣ ␣ subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.
Proceedings of The Physiological Society, 2003
Journal of Biological Chemistry, Mar 1, 2003
Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist... more Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1؉3.2A) that is activated by the direct binding of G␥ subunit to the channel complex to report, in real-time, using the patch clamp technique the activity of the "ternary complex" of agonist/receptor/G-protein. This analysis is further facilitated by the use of pertussis toxin-resistant fluorescent and non-fluorescent G␣ i/o subunits and a series of HEK293 cell lines stably expressing both channel and receptors (including the adenosine A 1 receptor, the adrenergic ␣ 2A receptor, the dopamine D 2S receptor, the M4 muscarinic receptor, and the dimeric GABA-B 1b/2 receptor). We systematically analyzed the contribution of the various inputs to the observed kinetic response of channel activation. Our studies indicate that the combination of agonist, GPCR, and G-protein isoform uniquely specify the behavior of these channels and thus support the importance of the whole ternary complex at a kinetic level.
American Journal of Physiology-cell Physiology, Jul 1, 2004
Biochemical Society Transactions, Oct 26, 2004
The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through h... more The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein α subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K + channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and G o α A-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gβγ overexpression. FRET was also seen between RGS8 and Gγ , demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein α subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the 'kinetic efficacy' of G-protein activation.
Scope: Contemporary Research Topics (Learning & Teaching)
Scope: Contemporary Research Topics (Learning and Teaching), 2021
An individual can't create anything itself. All of our dreams come true with the cooperation and ... more An individual can't create anything itself. All of our dreams come true with the cooperation and co-creation of other souls (Hashmi, 2014).
Proceedings of the Physiological Society, 2004
Proceedings of the National Academy of Sciences, 2005
Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric... more Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the α2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits α o , β1, and γ2 and a number of GPCRs including M4, α2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Gα proteins shows specificity: there is interaction between the α2A receptor and Go, but little interaction between the α2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heter...
Proceedings of the National Academy of Sciences, 2003
G protein-gated inwardly rectifying K + (Kir) channels are found in neurones, atrial myocytes, an... more G protein-gated inwardly rectifying K + (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G protein-coupled receptors (GPCRs) via the inhibitory family of G protein, G i/o , in a membrane-delimited fashion by the direct binding of Gβγ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K + channel, Kir3.1 + 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein α subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein α subunit. With som...
Journal of Biological Chemistry, 2005
Scope: Contemporary Research Topics (Learning and Teaching), 2021
Proceedings of the National Academy of Sciences of the United States of America, Apr 28, 2003
G protein-gated inwardly rectifying K ؉ (Kir) channels are found in neurones, atrial myocytes, an... more G protein-gated inwardly rectifying K ؉ (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G proteincoupled receptors (GPCRs) via the inhibitory family of G protein, G i/o, in a membrane-delimited fashion by the direct binding of G␥ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K ؉ channel, Kir3.1 ؉ 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein ␣ subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein ␣ subunit. With some combinations of agonist͞GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein ␣ subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs. G protein ␣ subunit ͉ inward rectifier ͉ cell signaling ͉ drug development ͉ G protein-signaling protein
Journal of Biological Chemistry, Aug 1, 2002
Signaling studies in living cells would be greatly facilitated by the development of functional f... more Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein ␣ subunits. We have designed G i/o ␣ subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-proteingated inwardly rectifying K ؉ channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G i/o ␣ subunits (G i1 ␣, G i2 ␣, G i3 ␣, and G oA ␣) and a mutation introduced at Cys ؊4 rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G i/o ␣ protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G i/o ␣ subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the G␥ dimer by the mutant G␣ subunit. Moreover, each subunit was able to support A1mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G i3 ␣ and G oA ␣ ␣ subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.
Proceedings of The Physiological Society, 2003
Journal of Biological Chemistry, Mar 1, 2003
Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist... more Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1؉3.2A) that is activated by the direct binding of G␥ subunit to the channel complex to report, in real-time, using the patch clamp technique the activity of the "ternary complex" of agonist/receptor/G-protein. This analysis is further facilitated by the use of pertussis toxin-resistant fluorescent and non-fluorescent G␣ i/o subunits and a series of HEK293 cell lines stably expressing both channel and receptors (including the adenosine A 1 receptor, the adrenergic ␣ 2A receptor, the dopamine D 2S receptor, the M4 muscarinic receptor, and the dimeric GABA-B 1b/2 receptor). We systematically analyzed the contribution of the various inputs to the observed kinetic response of channel activation. Our studies indicate that the combination of agonist, GPCR, and G-protein isoform uniquely specify the behavior of these channels and thus support the importance of the whole ternary complex at a kinetic level.
American Journal of Physiology-cell Physiology, Jul 1, 2004
Biochemical Society Transactions, Oct 26, 2004
The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through h... more The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein α subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K + channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and G o α A-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gβγ overexpression. FRET was also seen between RGS8 and Gγ , demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein α subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the 'kinetic efficacy' of G-protein activation.
Scope: Contemporary Research Topics (Learning & Teaching)
Scope: Contemporary Research Topics (Learning and Teaching), 2021
An individual can't create anything itself. All of our dreams come true with the cooperation and ... more An individual can't create anything itself. All of our dreams come true with the cooperation and co-creation of other souls (Hashmi, 2014).
Proceedings of the Physiological Society, 2004
Proceedings of the National Academy of Sciences, 2005
Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric... more Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the α2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits α o , β1, and γ2 and a number of GPCRs including M4, α2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Gα proteins shows specificity: there is interaction between the α2A receptor and Go, but little interaction between the α2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heter...
Proceedings of the National Academy of Sciences, 2003
G protein-gated inwardly rectifying K + (Kir) channels are found in neurones, atrial myocytes, an... more G protein-gated inwardly rectifying K + (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G protein-coupled receptors (GPCRs) via the inhibitory family of G protein, G i/o , in a membrane-delimited fashion by the direct binding of Gβγ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K + channel, Kir3.1 + 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein α subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein α subunit. With som...
Journal of Biological Chemistry, 2005