Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVβ-dependent manner

Abstract

Voltage-gated Ca²⁺ (Ca_V) channels couple membrane depolarization to Ca²⁺ influx, triggering a range of Ca²⁺-dependent cellular processes. Ca_V channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of Ca_V channels. GPCR-dependent mechanisms that downregulate Ca_V channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several Ca_V subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of Ca_V channels depends on the presence of a Ca_Vβ subunit. Ca_Vβ subunits displace Ca_Vα₁ subunits from the endoplasmic reticulum. The actions of GHSR on Ca_V channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.

Keywords: CaVβ; GPCR; Voltage-gated calcium (Ca2+) channels.

Fig. 1.

GHSR constitutive activity reduces Ca_V1.2 and Ca_V1.3 currents in tsA201 cells, and reduces native Ca_V1 currents in cultured hypothalamic neurons. (A) Representative Ca_V current traces from tsA201 cells co-transfected with Ca_V1.2, Ca_Vα₁δ₂, Ca_Vβ₃ and GHSR (+GHSR, n=8) or from controls transfected with empty plasmid (-GHSR, n=12), and average I_Ca for each condition (left). Representative Ca_V current traces from tsA201 cells co-transfected with Ca_V1.3, Ca_Vα₁δ₂, Ca_Vβ₃ and GHSR (+GHSR, n=13) pre-incubated or not with SPA 1 µM (+SPA, n=9) and from controls transfected with empty plasmid (-GHSR, n=19), and average I_Ca for each condition (right). (B) Representative traces of the Bay K 8644 (BayK) effect (5 µM) on the Ba²⁺ current from GHSR-deficient (GHSR null, n=5) and wild-type (Wild type, n=7) hypothalamic neurons (left), and average I_Ba increase (right). Error bars represent mean±s.e.m., individual points represents current registered (A) or current increase for each cell (B). Kruskal–Wallis with Dunn's post-test (Ca_V1.3), Mann–Whitney test (Ca_V1.2) (A), and Student's t-test (B).

Fig. 2.

GHSR constitutive activity fails to reduce Ca_V3.2 density on the plasma membrane. (A) Representative Ca_V current traces (left) from tsA201 cells co-transfected with Ca_V3.2 and GHSR (+GHSR, n=10) or empty plasmid (-GHSR, n=15), and average I_Ca for each condition (right). (B) Photomicrographs (left) and average of GFP plasma membrane signal (in percent) (right) of tsA201 cells co-transfected with Ca_V3.2-GFP and GHSR (+GHSR, n=88) and from controls transfected with empty plasmid (-GHSR, n=94). Green and red signals correspond to the eGFP tag on Ca_V3.2 and the CellMask membrane marker, respectively. Scale bar: 10 µm. Error bars represent mean±s.e.m., individual points represent each cell analyzed.

Fig. 3.

GHSR constitutive activity reduces Ca_V3.2 density on plasma membrane and, consequently, the Ca_V3.2 current in a Ca_Vβ₃-dependent manner. (A) Representative Ca_V current traces (left) from tsA201 cells co-transfected with Ca_V3.2, Ca_Vβ₃ and GHSR (+GHSR) pre-incubated or not with SPA 1 µM (+GHSR+SPA), and from controls transfected with empty plasmid (-GHSR) and average I_Ca in presence of increasing Ca_Vβ₃/Ca_V3.2 molar ratios (right). (B) Photomicrographs (left) and average of GFP plasma membrane signal (in percent) (right) of tsA201 cells co-transfected with Ca_V3.2-GFP, Ca_Vβ₃ and GHSR (+GHSR, n=21) pre-incubated or not with SPA 1 µM (+GHSR+SPA, n=14) and from controls transfected with empty plasmid (-GHSR, n=27). Green and red signals correspond to the eGFP tag on Ca_V3.2 and the membrane marker CellMask, respectively. Scale bar: 10 µm. Dots represent mean±s.e.m., numbers in brackets represent the number of analyzed cells in A. Error bars represent mean±s.e.m. and individual points represent each cell analyzed in B. Mann–Whitney test and Kruskal–Wallis with Dunn's post-test (A). Kruskal–Wallis with Dunn's post hoc test (B).

Fig. 4.

Presence of Ca_Vβ₃ but not that of Ca_Vα₂δ₁ is required for GHSR constitutive activity to reduce Ca_V2.2 current by decreasing Ca_V2.2 density on plasma membrane. (A) Representative Ca_V currents from tsA201 cells co-transfected with Ca_V2.2, Ca_Vα₂δ₁ and GHSR (+GHSR, n=44) or from controls transfected with empty plasmid (-GHSR, n=51) and average ICa for each condition. (B) Representative Ca_V currents from tsA201 cells co-transfected with Ca_V2.2, Ca_Vβ₃, Ca_Vα₂δ₁ and GHSR (+GHSR, n=22) or empty plasmid (-GHSR, 48) and average ICa for each condition. (C) Photomicrographs and averaged GFP plasma membrane signal (in percent) of tsA201 cells co-transfected with Ca_V2.2-GFP, Ca_Vα₂δ₁ and GHSR (+GHSR, n=87) and from controls transfected with empty plasmid (-GHSR, n=135) (top). Photomicrographs and average of GFP plasma membrane signal (in percent) of tsA201 cells co-transfected with Ca_V2.2-GFP, Ca_Vβ₃ and GHSR (+GHSR, n=51) or from controls transfected with empty plasmid (-GHSR, n=42) (middle). Photomicrographs and average of GFP plasma membrane signal (in percent) of tsA201 cells co-transfected with Ca_V2.2-GFP, Ca_Vβ₃, Ca_Vα₂δ₁ and GHSR (+GHSR, n=29) or from controls transfected with empty plasmid (-GHSR, n=27) (bottom). Green and red signals correspond to the eGFP tag on Ca_V2.2 and the membrane marker CellMask, respectively. Scale bars: 10 µm. Error bars represent mean±s.e.m. and individual points represent each cell analyzed. Mann–Whitney test.

Fig. 5.

GHSR constitutive activity reduces Ca_Vβ_2a density on the plasma membrane while increasing it on the ER. (A) Confocal images of tsA201 cells co-transfected with Ca_V2.2, Ca_Vβ_2a-eGFP, Ca_Vα₂δ₁, plasma membrane marker (PM-marker), endoplasmic reticulum-marker (ER-marker) and GHSR pre-incubated with SPA (1 µM) (+GHSR+SPA, n=18) or not (+GHSR, n=18) and controls expressing empty plasmid (-GHSR, n=19). (B) Confocal images of tsA201 cells co-transfected with Ca_V2.2, Ca_Vβ_2a-eGFP, Ca_Vα₂δ₁, Golgi complex marker (Golgi-marker), recycling endosome marker (RE-marker) and GHSR pre-incubated with SPA (1 µM) (+GHSR+SPA, n=15) or not (+GHSR, n=20) and controls expressing empty plasmid (-GHSR, n=16). Bar graphs show the colocalization of green signal from Ca_Vβ_2a-eGFP with blue signal (from PM-marker or Golgi-marker) or with red signal (from ER-marker or RE-marker). Error bars represent mean±s.e.m. and individual points represent each quantified cell. Scale bars: 10 µm. One way-ANOVA with Tukey's post hoc test (F_{2, 52}=10.28) (A) and Kruskal–Wallis with Dunn's post-test (B).

Fig. 6.

GHSR constitutive activity reduces Ca_Vβ₃ density on the plasma membrane while increasing it on the ER. (A) Confocal images of tsA201 cells co-transfected with Ca_V2.2, Ca_Vβ₃-eGFP, Ca_Vα₂δ₁, plasma membrane marker (PM-marker), endoplasmic reticulum-marker (ER-marker) and GHSR pre-incubated with SPA (1 µM) (+GHSR+SPA, n=14) or not (+GHSR, n=21) and of controls transfected with empty plasmid (-GHSR, n=25). (B) Confocal images of tsA201 cells co-transfected with Ca_V2.2, Ca_Vβ₃-eGFP, Ca_Vα₂δ₁, Golgi marker (Golgi-marker), recycling endosome marker (RE-marker) and GHSR pre-incubated with SPA (1 µM) (+GHSR+SPA, n=16) or not (+GHSR, n=16) and of controls transfected with empty plasmid (-GHSR, n=7). Bar graphs show the colocalization of green signal from Ca_Vβ₃-eGFP with blue signal (from PM-marker or Golgi-marker) or with red signal (from ER-marker or RE-marker). Scale bars:10 µm. Error bars represent mean±s.e.m., individual points represent each quantified cell. One way-ANOVA with Turkey's post-test (F_{2, 57}=16.43) (Ca_Vβ₃-eGFP/PM-marker overlap) and Kruskal–Wallis with Dunn's post-test.

Fig. 7.

GHSR constitutive activity fails to reduce Ca_Vβ density on plasma membrane in the absence of Ca_Vα₁. (A) Confocal images of tsA201 cells co-transfected with Ca_Vβ_2a-eGFP, Ca_Vα₂δ₁, plasma membrane marker (PM-marker), endoplasmic reticulum-marker (ER-marker) and GHSR (+GHSR, n=9) or of controls transfected with empty plasmid (-GHSR, n=9). (B) Confocal images of tsA201 cells co-transfected with Ca_Vβ₃-eGFP, Ca_Vα₂δ₁, plasma membrane marker (PM-marker), endoplasmic reticulum-marker (ER-marker) and GHSR (+GHSR, n=7) or of controls transfected with empty plasmid (-GHSR, n=8). Bar graphs show the colocalization of green signal from Ca_Vβ₃-eGFP or Ca_Vβ_2a-eGFP with blue signal (from PM-marker) or with red signal (from ER-marker). Scale bars: 10 µm. Error bars represent mean±s.e.m., individual points represent each quantified cell. Student's t-test (A) and Mann–Whitney test (B).

Fig. 8.

A W391A mutation of Ca_Vα₁ or truncation of Ca_Vβ are not sufficient to block the inhibitory effect of GHSR on Ca_V2.2 activity. (A) Representative Ca_V current traces from tsA201 cells co-transfected with Ca_V2.2W391A, Ca_Vα₁δ₂, Ca_Vβ₃ and GHSR (+GHSR, n=7) or from controls transfected with empty plasmid (-GHSR, n=7), and average I_Ca for each condition (top). Representative Ca_V current traces from tsA201 cells co-transfected with Ca_V2.2W391A, Ca_Vα₁δ₂, Ca_Vβ_2a and GHSR (+GHSR, n=5) or from controls transfected with empty plasmid (-GHSR, n=8), and average I_Ca for each condition (bottom). Error bars represent mean±s.e.m., individual points represents current registered. Student's t-test (top) and Mann–Whitney test (bottom). (B) Representative Ca_V current traces from tsA201 cells co-transfected with Ca_V2.2, Ca_Vα₁δ₂, Ca_Vβ_2aTF8n and GHSR (+GHSR, n=10) or from controls transfected with empty plasmid (-GHSR, n=14), and average I_Ca for each condition. Error bars represent mean±s.e.m., individual points represents current registered. Mann–Whitney test.