rSK1 in Rat Neurons: A Controller of Membrane rSK2?

Figures

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  Figure 1. rSK1-KD 3.6 reduces rSK1-YFP expression in HEK293T cells. Western blot of total cell lysates of HEK293T cells transfected with rSK1-YFP and co-transfected with pcDNA3 or plldsRed or three different knock-down hairpin constructs: rSK1-KD 2.4 or rSK1-KD 3.6 or rSK1-KD 4.5. rSK1-KD 3.6 reduced the expression of rSK1-YFP by over 90% and was used in all further experiments. β-actin was used as a loading control.

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  Figure 2. rSK1 regulates the membrane expression of rSK2 channels. (A) Immunocytochemistry of Cosm6 cells transfected with rSK2-Myc or rSK1-HA. rSK2-Myc (pink), rSK1-HA (green), DAPI (blue). (B) Immunoblots from Cosm6 cells transfected with rSK1-HA, rSK2-Myc and hSK1 in different combinations. Shown are the total lysate (LYS) and membrane (MEMB) fraction isolated using surface biotinylation probed with either Myc (rSK2; left) or HA (rSK1; right) antibody. EGFR was used as the loading control. rSK2 transfection results in three size bands that represent monomeric protein (band 3, ~60 kDa), dimers (band 2, ~120 kDa) and tetramers (band 1, >200 kDa). Co-transfection of rSK2 and rSK1 reduced the amount of rSK2 expressed in the cell membrane. (C) Bar graphs show normalized relative density of SK2, as described in the “Materials and Methods” section, in the membrane fraction from Cosm6 cells transfected with rSK2 alone or co-transfected with rSK1 or hSK1. Co-transfection with rSK1-HA significantly reduced the 60 kDa band (p = 0.002). Co-transfection with hSK1 increased expression of rSK2. (D) Co-immunoprecipitation (co-IP) assay of transfected Cosm6 cells shows co-assembly of rSK1-rSK2.…

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  Figure 3. rSK1 and rSK2 expressed in rat hippocampal neuronal cultures. Cultured hippocampal neurons were transduced with rSK2-Myc (A) or rSK1-HA (B) or both (C) using lentivirus and immunostained for DAPI (blue), the neuronal marker NeuN (red), Myc (purple) or HA (green). (A,B) SK2 (Myc) and SK1 (HA) are expressed throughout the soma and processes of transduced neurons. DAPI (C) co-expression of rSK2 and rSK1 shows that both proteins are present in the same compartments. (D) Immunocytochemistry using a specific SK2 antibody (c-39; green) shows that endogenous rSK2 is present in the soma and throughout the processes of cultured neurons. Hippocampal neurons are marked using CaMKII (purple).

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  Figure 4. rSK1 regulates membrane expression of rSK2 channels in cultured neurons. Cultured hippocampal neurons were transduced to express rSK1-HA or rSK2-Myc or both. Surface protein was biotinylated, and solubilized protein from neurons separated into total lysate (LYS) and membrane fraction (MEMB) as detailed in the “Materials and Methods” section. Immunoblots were probed using the Myc antibody to test for rSK2 expression. Na/K ATPase or β-actin was used as loading controls. (A) Co-expression of rSK1 and rSK2 reduces membrane expression of rSK2. (B) Graph shows normalized relative density of SK2, in the membrane fraction from neurons transduced with rSK2 alone or rSK2 and rSK1. Co-infection significantly reduced the band at 200 kDa (p = 0.04). (C) Expression of rSK1 in neurons reduced surface expression of endogenous rSK2. Cultured neurons were transduced to express rSK1 using lentivirus. Blots show solubilized protein from the membrane fraction (upper blots) and cytosolic fraction (lower blots) from control neurons (left) and neurons transduced with rSK1(right). Blots were probed with anti SK2 antibody (c-39). (D) Quantification of SK2 protein expression in the membrane (upper graph)…

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  Figure 5. Knock down of rSK1 enhances IAHP in hippocampal neurons. Cultured hippocampal neurons were transduced to express a hairpin knockdown vector for SK1 (rSK1-KD) or a scrambled control vector (SCRAM). Surface protein was biotinylated, and solubilized protein from neurons separated into total lysate (LYS) and membrane fraction (MEMB) as detailed in the “Materials and Methods” section. Blots were immunoprobed using the rSK2 antibody (c-39). (A1) Knockdown of SK1 protein results in an overall reduction of total endogenous monomeric SK2 protein, but enhancement of high molecular weight SK channels in the plasma membrane. Na+/K+ ATPase was sued as the loading control. (A2) Normalized relative density of endogenous rSK2 in the membrane fraction [NORM (MEMB)] from neurons infected with rSK1-KD. rSK1-KD significantly decreased the amount of endogenous monomeric rSK2, but increased high molecular weight rSK2. Na+/K+ ATPase (~110 kDa) was used to normalize all the loaded samples for the MEMB fraction. (B1) Knockdown of rSK1 results in a reduction in high molecular weight SK2 in the cytoplasmic fraction. (B2) Quantification of blot shown in B1. β-actin (~40 kDa) was used to normalize all the…