Ion channels in the regulation of autophagy

<p>Autophagy is a cellular process in which the cell degrades and recycles its own constituents. Given the crucial role of autophagy in physiology, deregulation of autophagic machinery is associated with various diseases. Hence, a thorough understanding of autophagy regulatory mechanisms is crucially important for the elaboration of efficient treatments for different diseases. Recently, ion channels, mediating ion fluxes across cellular membranes, have emerged as important regulators of both basal and induced autophagy. However, the mechanisms by which specific ion channels regulate autophagy are still poorly understood, thus underscoring the need for further research in this field. Here we discuss the involvement of major types of ion channels in autophagy regulation.</p

Effect of deletions on channel surface expression as measured by electrophysiology and luminometry.

<p><i>A,B,</i> The <i>I-V</i> data from each cell was fit with a Boltzmann-Ohm equation to calculate <i>V_0.5</i>, <i>k</i>, <i>G_max</i>, and <i>E_rev</i>. The value of <i>E_rev</i> was then used to calculate the chord conductance at each test potential. These data were then normalized for cell capacitance. Only the increase in GD3–5 conductance (G_max) was statistically different than control Ca_v3.1. <i>C,</i> Luminometric quantification of the expression levels of HA-tagged Ca_v3.1 and Ca_v3.3 channel variants at the membrane (non-permeabilized). Average relative light units (RLU) for WT channels before normalization were: Ca_v3.1, 2,867,417±551,473; and Ca_v3.2 2,533,150±353,578, n = 6 for both). To reduce the error between experiments the data were normalized to the membrane expression for the respective WT channel. Total expression of HA-tagged channels measured after Triton X-100 permeabilization normalized to membrane expression. Ratio of surface/total expression identifies an increase in membrane expression for GD3–5, and decreases in GD1–2, ID1–2, and ID3–5. Statistically significant differences from control wild-type channels are indicated (**p<0.01; ***p<0.001).</p

Effect of Ca_v3.1 I–II loop deletions on the voltage dependence of channel activation.

<p><i>A, B,</i> Normalized current traces recorded during depolarizing voltage steps from −80 to +30 mV (holding potential, −100 mV, except for GD1–2 mutant, which due to shifted inactivation was −110 mV) in WT Ca_v3.1 (<i>Aa</i>), GD1–2 (<i>Ab</i>), GD3–5 (<i>Ac</i>), WT Ca_v3.3 (<i>Ba</i>), ID1–2 (<i>Bb</i>) and ID3–5 (<i>Bc</i>). Thick gray lines represent the current at −50 mV, demonstrating the negative shift in voltage dependence of activation observed in the deletion mutants. Currents were normalized to the maximum peak current in that cell. Time calibration bar scale applies to all three sets of traces in each case. Peak current-voltage plots for either Ca_v3.1 and its deletions (<i>C</i>) or Ca_v3.3 and its deletions (<i>D</i>). Peak currents were normalized to the cell size as estimated by capacitance. Normalized current-voltage plots for either Ca_v3.1 and its deletions (<i>E</i>) or Ca_v3.3 and its deletions (<i>F</i>). Same symbol definition as in panels <i>C</i> and <i>D</i>. Smooth curves in <i>C–F</i> represent fits to the average data using a Boltzmann–Ohm equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002976#pone.0002976-AriasOlgun1" target="_blank">[4]</a>.</p

Effect of D1–2 deletions on kinetics of Ca_v3.1 and Ca_v3.3 kinetics.

<p><i>A</i>, <i>B,</i> Normalized current traces for Ca_v3.1 (thick gray line), Ca_v3.3 (dashed line), and ID1–2. Currents were recorded during step depolarizations to −10 mV. The same current traces are shown in <i>A</i> and <i>B</i>, but at a different time scale. In <i>A</i> the time scale is expanded to illustrate how ID1–2 activates as fast as Ca_v3.1, while in <i>B</i> a longer time scale is shown to illustrate how ID1–2 inactivates at a similar rate as WT Ca_v3.3. <i>C,</i> Average activation kinetics estimated using a 2 exponential fit to the raw current traces obtained during the <i>I-V</i> protocol. Data represent mean±s.e.m , and N is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002976#pone-0002976-t001" target="_blank">Table 1</a>. <i>D,</i> Average inactivation kinetics. Same symbol definition as in panel <i>C</i>.</p

Estimating the effect of the deletions on the probability of channel opening.

<p><i>Aa</i>, Schematic of the P/-8 voltage protocol. <i>Ab</i>, Representative current at full scale, which is expanded in panel <i>Ac</i>. <i>B,C</i> Representative gating current traces recorded during depolarizing voltage steps from −100 to ∼ +50 mV (reversal potential): WT Ca_v3.1 (<i>Ba</i>); GD1–2 (<i>Bb</i>); GD3–5 (<i>Bc</i>); WT Ca_v3.3 (<i>Ca</i>); ID1–2 (<i>Cb</i>); and ID3–5 (<i>Cc</i>). Vertical scale bar is same size for all six traces (0.1 nA), while the horizontal scale bar is 1 ms in <i>B</i> and 2 ms in <i>C</i>. Data were acquired at 20 kHz, filtered at 10 kHz, and represent the average of 20 runs. G_max vs. Q_max for WT Ca_v3.1 and GD1–2 (<i>D</i>), or WT Ca_v3.3 and ID1–2 (<i>E</i>). The slope of the linear regression fit provides an estimate of <i>P_o,</i> and in both cases the slope of the line fitting the D1–2 mutants was 2-fold higher than for WT (Ca_v3.1, 0.26±0.03, n = 9; GD1–2, 0.55±0.06, n = 6, P<0.001; Ca_v3.3, 0.12±0.01, n = 9; and ID1–2, 0.26±0.02, n = 6, P<0.05). The difference between Ca_v3.1 and Ca_v3.3 is also statistically significant (P<0.001, one-way ANOVA followed by Tukey's multiple comparison test, Prism).</p

Electrophysiological properties of Ca_v3.1, Ca_v3.3, and their deletion mutants.

<p>The <i>G_max</i> and <i>V_0.5</i> of activation were determined from the <i>I-V</i> protocol, and therefore have the same number of cells (n) in each measurement. The <i>G/Q</i> ratio was calculated for each individual cell, and then averaged. Statistical significance is denoted with asterisks, where three asterisks indicates P<0.001, two for P<0.01, and one for P<0.05.</p

Location of deletions in the I–II loop.

<p>(<i>A</i>), Schematic representation of the I–II loop connecting repeat IS6 to repeat IIS1 in Ca_v3.1 and Ca_v3.3. Deleted regions are shown as open boxes. (<i>B</i>), Alignment of the I–II loop of human Ca_v3 channels. Arrows indicate where deletions begin and end. Dashes represent gaps in the alignment. Amino acids are color-coded by their physical properties as follows: red, positively-charged; green, negatively-charged; blue, polar; and yellow, hydrophobic.</p

Primers and siRNA.

<p>Primers and siRNA.</p

The effects of 1,25-dihydroxyvitamin D3 on androgen-independent cell lines.

<p><b>A</b>, <b>B</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen receptor-deficient DU-145 cell line in both 2 and 10% FCS-containing RPMI medium (<b>A</b> and <b>B</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>C, D</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen-insensitive LNCaP C4-2 cell line in both 2 and 10% FCS-containing RPMI medium (<b>C</b> and <b>D</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>E</b>, the relative expression levels of TRPV6 channel in DU-145 cells treated with 100 µM 1,25-dihydroxyvitamin D3 for 3 days in 2 and 10% FCS-containing RPMI medium, * - P<0.05 (as compared to control), n = 3. <b>F</b>, the expression of TRPV6 channel induced by 100 nM 1,25-dihydroxyvitamin D3 for 3 days in LNCaP cells in steroid-deprived RPMI medium (LNCaP-ST), n = 3.</p

The effects of 1,25-dihydroxyvitamin D3 on proliferation and apoptosis resistance of LNCaP cells are mediated via TRPV6 channel.

<p><b>A</b>, LNCaP cells proliferation in 2% FCS-containing RPMI medium treated with 1,25-dihydroxyvitamin D3 (100 nM, applied at D1), siRNA-TRPV6 (siTRPV6, 80 nM, transfected at D0), the combined treatment of 1,25-dihydroxyvitamin D3 and siTRPV6 specified above, and siRNA-AR (siAR, 80 nM, transfected at D0) as a positive control. * - P<0.05, ** - P<0.01, as compared to control, n = 4; <b>B</b>, a cell cycle assay of LNCaP cells (incubated with 2% FCS-containing RPMI medium) for the same conditions as in MTS assay (<b>A</b>) (D3 equals 100 nM 1,25-dihydroxyvitamin D3), carried out by flow cytometry of the cells stained with propidium iodide. * - P<0.05, ** - P<0.01, § - P<0.05 vs. Vitamin D3; n = 3. <b>C</b>, a western-blotting of proliferating cell nuclear antigen (PCNA) in the conditions indicated above as compared to β-actin. <b>D</b>, an apoptosis assay carried out by flow cytometry as a subG1 population of LNCaP cells cultured in 2% FCS-containing RPMI medium stained with propidium iodide. * - P<0.01 vs. control; n = 3.</p