Figure 2
From: Aldosterone up-regulates voltage-gated potassium currents and NKCC1 protein membrane fractions
taken from the same gel. (c) Whole cell voltage-clamp recordings of a representative SH-SY5Y show outward currents elicited by 300 ms voltage steps. Voltage steps from -60 mV to + 30 mV, in 10 mV step increments were acquired at a holding potential was −90 mV. There was a reversible decrease in K+ currents due to application of tetraethyl ammonium – TEA (1 mM), confirming the presence of K+ channels in SH-SY5Y cells. (d) For a representative cell, current traces at + 30 mV voltage step shows the effects of TEA on K + currents. (e) Similarly, mean reduction in currents with respect to control (n = 6) was shown when cells were perfused with 1 mM TEA. (f) There was a reversible increase in potassium currents when 1 μM ALD was applied as compared to no ALD and currents decreased to the control values when ALD was washed out. (g) Mean I–V functions are plotted for control (n = 17 cells) and after ALD application (n = 5 for wash) were shown as mean current–voltage relations. (h) The change in outward currents with respect to control conditions following ALD application were greater for more positive holding potentials. (i) To further confirm the increase in K+ due to ALD application, ALD was perfused in combination with the K+ blocker, TEA. Currents traces of a representative cell were shown as outward currents in response to test potentials −60 mV to + 30 mV, in 10 mV increments with holding potential −90 mV. There was an increase in potassium currents due to ALD, followed by decrease in currents when TEA was added to the bath. Currents return to control values during wash out. (j, k) Current traces at + 30 mV testing voltage and mean change with respect to control values in currents confirm the same tendency (n = 5). Statistical Significance: *P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001.
Aldosterone induces increases in NKCC1 protein expression levels and outward potassium current amplitudes. (a) SH-SY5Y cells were treated with aldosterone (ALD) at concentrations from 1 nM to 10 μM for 24 h. Cell lysates were performed in PBS buffer and membrane fractions were separated using the cell signaling kit. Western blot analysis probed with NKCC1 and β-actin antibodies for both total lysate and membrane fractions. β-actin was used as the loading control and equal amounts of lysate protein were used for the isolation of cell membrane proteins (lysate protein concentration was measured by spectrophotometer). For both total lysate and membrane fractions, NKCC1 protein expression was upregulated following application of ALD as compared to control samples (no ALD treatment). There was no β-actin present in the membrane fractions as it is a structural protein. Data are means ± SD from 3 independent experiments. (b) NKCC1 mRNA was not regulated by ALD treatment. Cell lysates performed in RLT buffer and total RNA was isolated using the Qiagen kit. Subsequently, real-time RT-PCR technique was carried out. There was no significant difference between the control (no ALD) and treated samples (n = 3). All western blot data were
