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Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons.

López Soto EJ, Agosti F, Cabral A, Mustafa ER, Damonte VM, Gandini MA, Rodríguez S, Castrogiovanni D, Felix R, Perelló M, Raingo J - J. Gen. Physiol. (2015)

Bottom Line: Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear.Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway.Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Electrophysiology and Laboratory of Neurophysiology, Multidisciplinary Institute of Cell Biology (IMBICE), B1904CMA La Plata, Buenos Aires, Argentina.

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GHSR1a decreases surface CaV2.1 and CaV2.2 density. (A) Photomicrographs and averaged percentages of green fluorescent plasma membrane signal of HEK 293T cells transfected with CaV2.1-GFP (left) and CaV2.2-GFP (right), its auxiliary subunits (Control) with GHSR1a or GHSR1a-A204E, and preincubated or not with 1 µM SPA or 500 ng/ml PTx. Green and red signals correspond to the eGFP tag on CaV2 and the membrane marker CellMask, respectively. Kruskal–Wallis with Dunn’s post-test; *, P < 0.01. (B) Western blot analysis displaying the CaV2.1-GFP and CaV2.2-GFP protein level in the plasma membrane (PM) or the cytoplasmic (Cyt) fraction of HEK 293T cells transfected with CaV2.1-GFP or CaV2.2-GFP and its auxiliary subunits (Control), and cotransfected with GHSR1a or GHSR1a-A204E (left), and averaged percentage of CaV2.1-GFP and CaV2.2-GFP PM protein level in each condition normalized against cadherin signal used as the plasma membrane loading control (right). Both AKT and Hsp90 as cytosolic markers. n = 2 and 3 for CaV2.1-GFP or CaV2.2-GFP, respectively. Error bars represent mean ± SE.
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fig4: GHSR1a decreases surface CaV2.1 and CaV2.2 density. (A) Photomicrographs and averaged percentages of green fluorescent plasma membrane signal of HEK 293T cells transfected with CaV2.1-GFP (left) and CaV2.2-GFP (right), its auxiliary subunits (Control) with GHSR1a or GHSR1a-A204E, and preincubated or not with 1 µM SPA or 500 ng/ml PTx. Green and red signals correspond to the eGFP tag on CaV2 and the membrane marker CellMask, respectively. Kruskal–Wallis with Dunn’s post-test; *, P < 0.01. (B) Western blot analysis displaying the CaV2.1-GFP and CaV2.2-GFP protein level in the plasma membrane (PM) or the cytoplasmic (Cyt) fraction of HEK 293T cells transfected with CaV2.1-GFP or CaV2.2-GFP and its auxiliary subunits (Control), and cotransfected with GHSR1a or GHSR1a-A204E (left), and averaged percentage of CaV2.1-GFP and CaV2.2-GFP PM protein level in each condition normalized against cadherin signal used as the plasma membrane loading control (right). Both AKT and Hsp90 as cytosolic markers. n = 2 and 3 for CaV2.1-GFP or CaV2.2-GFP, respectively. Error bars represent mean ± SE.

Mentions: Based on our results showing that the GHSR1a inverse agonist, SPA, requires long preincubation periods to occlude constitutive inhibition of CaV2 by GHSR1a, we decided to test if surface CaV2 density was affected by constitutive GHSR1a activity. First, we monitored the presence of CaV2 channels in the plasma membrane, using CaV2.1 and CaV2.2 channels tagged with GFP (CaV2.1-GFP and CaV2.2-GFP) that we have confirmed are functional in our experimental conditions (CaV2.1-GFP current, −47.0 ± 14.5 pA/pF, n = 9; CaV2.2-GFP current, −51.8 ± 12.3 pA/pF, n = 5). To identify the surface location of the GFP fluorescence signal, we used the red fluorescent membrane marker CellMask (Cogger et al., 2010). We found that CaV2.1-GFP– and CaV2.2-GFP–associated fluorescence signal was significantly lower in cells coexpressing GHSR1a, as compared with those coexpressing GHSR1a-A204E or those coexpressing GHSR1a and preincubated with SPA or PTx (Fig. 4 A). Next, we used Western blots to confirm that the CaV2.1 and CaV2.2 membrane protein level is decreased when cells coexpress GHSR1a. We used HEK 293T cells transfected with CaV2.1 or CaV2.2, as well as GHSR1a, GHSR1a-A204E, or the pcDNA3.1 empty vector. By using cadherin as a plasma membrane marker, and AKT and Hsp90 as cytoplasmic protein markers, we found that CaV2.1 and CaV2.2 protein quantity decreased in the plasma membrane protein fraction when cells coexpress GHSR1a, whereas GHSR1a-A204E coexpression failed to affect the amount of CaV2.1 and CaV2.2 plasma membrane protein (Fig. 4 B). Collectively, our data suggest that constitutive GHSR1a activity reduces surface expression of CaV2.1 and CaV2.2 channels by a Gi/o-dependent mechanism.


Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons.

López Soto EJ, Agosti F, Cabral A, Mustafa ER, Damonte VM, Gandini MA, Rodríguez S, Castrogiovanni D, Felix R, Perelló M, Raingo J - J. Gen. Physiol. (2015)

GHSR1a decreases surface CaV2.1 and CaV2.2 density. (A) Photomicrographs and averaged percentages of green fluorescent plasma membrane signal of HEK 293T cells transfected with CaV2.1-GFP (left) and CaV2.2-GFP (right), its auxiliary subunits (Control) with GHSR1a or GHSR1a-A204E, and preincubated or not with 1 µM SPA or 500 ng/ml PTx. Green and red signals correspond to the eGFP tag on CaV2 and the membrane marker CellMask, respectively. Kruskal–Wallis with Dunn’s post-test; *, P < 0.01. (B) Western blot analysis displaying the CaV2.1-GFP and CaV2.2-GFP protein level in the plasma membrane (PM) or the cytoplasmic (Cyt) fraction of HEK 293T cells transfected with CaV2.1-GFP or CaV2.2-GFP and its auxiliary subunits (Control), and cotransfected with GHSR1a or GHSR1a-A204E (left), and averaged percentage of CaV2.1-GFP and CaV2.2-GFP PM protein level in each condition normalized against cadherin signal used as the plasma membrane loading control (right). Both AKT and Hsp90 as cytosolic markers. n = 2 and 3 for CaV2.1-GFP or CaV2.2-GFP, respectively. Error bars represent mean ± SE.
© Copyright Policy - openaccess
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fig4: GHSR1a decreases surface CaV2.1 and CaV2.2 density. (A) Photomicrographs and averaged percentages of green fluorescent plasma membrane signal of HEK 293T cells transfected with CaV2.1-GFP (left) and CaV2.2-GFP (right), its auxiliary subunits (Control) with GHSR1a or GHSR1a-A204E, and preincubated or not with 1 µM SPA or 500 ng/ml PTx. Green and red signals correspond to the eGFP tag on CaV2 and the membrane marker CellMask, respectively. Kruskal–Wallis with Dunn’s post-test; *, P < 0.01. (B) Western blot analysis displaying the CaV2.1-GFP and CaV2.2-GFP protein level in the plasma membrane (PM) or the cytoplasmic (Cyt) fraction of HEK 293T cells transfected with CaV2.1-GFP or CaV2.2-GFP and its auxiliary subunits (Control), and cotransfected with GHSR1a or GHSR1a-A204E (left), and averaged percentage of CaV2.1-GFP and CaV2.2-GFP PM protein level in each condition normalized against cadherin signal used as the plasma membrane loading control (right). Both AKT and Hsp90 as cytosolic markers. n = 2 and 3 for CaV2.1-GFP or CaV2.2-GFP, respectively. Error bars represent mean ± SE.
Mentions: Based on our results showing that the GHSR1a inverse agonist, SPA, requires long preincubation periods to occlude constitutive inhibition of CaV2 by GHSR1a, we decided to test if surface CaV2 density was affected by constitutive GHSR1a activity. First, we monitored the presence of CaV2 channels in the plasma membrane, using CaV2.1 and CaV2.2 channels tagged with GFP (CaV2.1-GFP and CaV2.2-GFP) that we have confirmed are functional in our experimental conditions (CaV2.1-GFP current, −47.0 ± 14.5 pA/pF, n = 9; CaV2.2-GFP current, −51.8 ± 12.3 pA/pF, n = 5). To identify the surface location of the GFP fluorescence signal, we used the red fluorescent membrane marker CellMask (Cogger et al., 2010). We found that CaV2.1-GFP– and CaV2.2-GFP–associated fluorescence signal was significantly lower in cells coexpressing GHSR1a, as compared with those coexpressing GHSR1a-A204E or those coexpressing GHSR1a and preincubated with SPA or PTx (Fig. 4 A). Next, we used Western blots to confirm that the CaV2.1 and CaV2.2 membrane protein level is decreased when cells coexpress GHSR1a. We used HEK 293T cells transfected with CaV2.1 or CaV2.2, as well as GHSR1a, GHSR1a-A204E, or the pcDNA3.1 empty vector. By using cadherin as a plasma membrane marker, and AKT and Hsp90 as cytoplasmic protein markers, we found that CaV2.1 and CaV2.2 protein quantity decreased in the plasma membrane protein fraction when cells coexpress GHSR1a, whereas GHSR1a-A204E coexpression failed to affect the amount of CaV2.1 and CaV2.2 plasma membrane protein (Fig. 4 B). Collectively, our data suggest that constitutive GHSR1a activity reduces surface expression of CaV2.1 and CaV2.2 channels by a Gi/o-dependent mechanism.

Bottom Line: Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear.Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway.Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Electrophysiology and Laboratory of Neurophysiology, Multidisciplinary Institute of Cell Biology (IMBICE), B1904CMA La Plata, Buenos Aires, Argentina.

Show MeSH
Related in: MedlinePlus