Skip to main content
. 2019 Apr 3;13:21. doi: 10.3389/fncir.2019.00021

Figure 5.

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 loaded samples for the CYTO fraction. (C) Knockdown of rSK1 in hippocampal neurons (red traces) increased the amplitude of the IAHP in voltage clamp (left) and the medium AHP in current clamp (right) in comparison to SCRAM infected neurons (traces in gray). (D) Quantification of the impact of the increase in the IAHP current shown as pA/pF and the medium AHP following knockdown of rSK1. (E) The enhanced IAHP current following knockdown of rSK1 is apamin (100 nM) sensitive consistent with SK2 channels.