Functional characterization of recombinant K_V1.1 homo-tetramers reveals distinctive biophysical profiles from those of K_V1.1/1.2 heteromers.

<p>(<b>A</b>) Western blots of surface expressed concatenated K_V1 channels in[HEK293 cells. Lanes: 1, non-transfected cells show no immuno-reactivity for K_V1.1 (or K_V1.2, not shown); 2, K_V(1.1)₄ and 3, K_V1.1-1.1-1.2-1.1 detected with anti-K_V1.1 IgG giving a band size of ∼250 kD; 4 and 6, K_V(1.2)₄ homo-tetramer was non-reactive with anti-K_V1.1 IgG (4) but gave a distinct band when probed with K_V1.2 IgG (6). Protein markers are indicated in lanes 5 and 7. (<b>B, D1–F1</b>) Representative recordings of macroscopic currents (300 ms pulse) from HEK293 cells transfected with the individual recombinant channels. (<b>B, C</b>) Activation rate of the voltage-dependent K⁺ currents mediated by K_V(1.1)₄ (left) and K_V(1.2)₄ (middle) channels (within the range of 10–30% of max. current) at 5 mV from indicated voltages (below) with super-imposed (right) representative traces from. A notable difference between the rates of activation of K_V(1.1)₄ and K_V(1.2)₄ is revealed by fitting the data with a single exponential (see <b>C</b>). (<b>D2–F2</b>) Conductance-voltage relations of macroscopic currents measured, based on the K⁺ current of the last 100 ms for each channel. Conductance at various command potentials were normalised and fitted with a single Boltzmann function. The difference in conductance values of K_V(1.1)₄ and K_V(1.2)₄ channel were statistically significant from −55 mV (P<0.05, Mann-Whitney <i>U</i>-test, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087736#pone-0087736-t001" target="_blank">Table 1</a> for summary of the biophysical data).</p

Demyelination disrupts the conductivity of ON axons which can be partially restored by 4-AP.

<p>(A, B) A low magnification micrograph (4×) demonstrating the semi-dissected ON (ventral view) with stimulation (suction, Suc. pipette) and recording (Rec.) electrodes. Graded synchronous CAPs recorded from control animals contrasting with bi-component CAPs derived from experimental ON activated from elevated stimuli thresholds (<b>C</b>). Insert illustrates the experimental set-up for CAPs recordings. Rec. - recording electrode; Suc. - suction pipette used for stimulation. ON – optic nerve, OX – optic xiasm. (<b>B</b>) Typical CAPs evoked in control ON by paired-pulse stimulation (PPS). Note the second CAP from the refractory phase following the first CAP. The evoked CAPs recorded from cuprizone-treated (demyelinated) ON axons showed lower amplitudes and protracted late components compared to the untreated (myelinated) ON axons. (<b>C</b>) Stimulus-response relation of CAPs in controls and experimental ON, showing lower activation threshold and higher amplitudes of evoked CAPs in demyelianted ON. (<b>D</b>) Representative recordings of CAPs from ON of control and cuprizone fed mice before (1) in the presence of TEA (2, upper row) or 4-AP (lower row) and (1+2) superimposed traces. (F, G) Summary of the effects of TEA (15–20 min application) on the CAPs in control and cuprizone-treated ONs (n = 5 in each group) (<b>E</b>) The summary histogram of CAP amplitudes scored before and after application of 1 mM 4-AP. Note the significant enhancement of the CAP amplitudes in demyelinated ON caused by 4- AP (P<0.05, n = 5 in each group).</p

V_½ for activation and onset rate of currents mediated by the different recombinant channels expressed in HEK293 cells.

<p>Results are represented as means ±S.E.M. (n-values);</p>*<p>(p<0.05) and ** (p<0.005) numbers are significant compared to those from K_V(1.1)₄, (Mann Whitney <i>U</i>-test);</p>#<p>data are taken from Al-Sabi et al., (2010).</p

Demyelination alters the distribution and composition of K_V1 channels in ON.

<p>Double [pan-Na (red)/K_V1.2 (green)] immuno-labelling of control (<b>A1</b>) and experimental (<b>A2</b>) ON: note elongated JXPs with alterations in most of the nodal Na_V channel clusters in samples from the cuprizone-treated mice. (<b>B1–2</b>) Double immuno-labelling of ON for K_V1.1 (red) and K_V1.2 (green) subunits of K_V1 channels: control (<b>B1</b>) and experimental (<b>B2</b>) samples, respectively. Note the highly localized occurrence of these proteins in JXPs of controls contrasting with their diffuse location along the ON axons in demyelinated specimens. Yellow staining corresponds to JXP regions showing co-localization of these proteins. The scale bars for low and high magnifications are 6 and 2 µm, respectively. (<b>C</b>) Summary histogram of the intact JXP labelled with anti-K_V1.2 antibody of control and experimental ON axons (n = 3 in each group). (<b>D</b>) A plot of the mean area of JXPs labelled for K_V1.1 channels in control (2.4±0.5 µm²) compared to the increased area of fluorescence intensity of JXPs in demyelinated (8.2±1 µm²) axons. (<b>E</b>) The mean fluorescence area of JXPs labelled for Kv1.2 channels in control (3.8±0.4) was lower than that in the treated ON axons (8.2±1 µm²). (<b>F</b>) A summary histogram of K_V1.1 and 1.2 co-localization in control (0.86±0.06) and demyelinated (0.27±0.04) ON demonstrating a significant (p<0.001) reduction in the degree of K_V1.1/1.2 co-localization in ON axons of the experimental mice. (<b>G</b>) The degree of K_V1.2/1.1 co-localization in ON axons of the experimental mice showed a reduction, which is still significant (P<0.05), when comparing the control (0.71±0.06) and the demyelinated ON (0.49±0.04) values. Data are taken from control and demylinated ON axons of 3 animals, in each group.</p

Differential inhibition of concatenated K_V1 channels expressed in HEK293 cells by DTX_K and TsTX-Kα.

<p>Results are represented as means ±S.E.M.; n-values are in brackets;</p>*<p>(p<0.05) numbers are significant compared to K_V(1.1)₄ (<i>t</i>-test),</p>#<p>Data were taken from Al-Sabi et al., (2010).</p

Cuprizone administration to mice induces widespread demyelination in several brain structures and reduces the myelin content.

<p>(<b>A–B</b>) Representative light micrographs of sagittal sections of brain from control and cuprizone-treated mice, respectively, stained for myelin with CV and LFB dyes. (<b>A</b>) Myelin is visualised in samples from the control mice, as light brown by CV and dense blue for LFB, in corpus callosum (CC), the stripes in the corpus striatum of the caudate putamen (CPu), internal capsule (IC) and the central nuclei of the cerebellar medulla (CN). (<b>B</b>) Extensive demyelination was observed in the above regions of treated mice. (<b>C</b>) Plot of the density distribution of myelin stained with LFB of defined colossal ROIs. A narrow and leftward-shift in the histogram of the density of myelin in cuprizone-treated samples reflects callosal demyelination (n = 6 in each group). (<b>D</b>) Correlation analysis of the density of LFB-stained myelin of random ROIs from callosal and hippocampal CA3 areas. Note the higher level of callosal myelin in controls (R² = 0.21) compared to CA3 area, with its stronger decline in experimental samples (R² = 0.54). (<b>E</b>) Summary histogram of the mean myelin density in CC and CP regions (>100 ROIs, from each group) with both areas revealing significant loss of myelin in mice that had received cuprizone. * p<0.05.</p

A more promenant contribution of K_V1.1 than 1.2 subunits of K_v channels in regulating the excitability and conductivity of demyelinated ON.

<p>(<b>A1, B1</b>) Representative CAP recordings demonstrating the effects of DTX_K and TsTX-Kα respectively, before (1) and 40 min after presentation of the toxins to the control and experimental ONs (2). (<b>A2, B2</b>) Time course of the effects of DTX_K and TsTX-Kα, respectively, on evoked CAPs (sub-maximal) of control (filled circle; n = 5; n = 6) and experimental (open circle; n = 5; n = 5), respectively. Black arrows indicate the start of the application of toxins. Note a slight increase in the CAPs by these toxin blockers in controls (A1, B1, filled circle) compared to much stronger enhancement of CAPs in demyelinated ON by TsTX-Kα (B1 and B2) and especially DTX_K (A1 and A2).</p

Light- and electron-microscopic analysis of ON reveals a decrease in the compactness and loss of myelin in the experimental mice.

<p>(<b>A</b>) Low power representative photomicrographs of ON from control and cuprizone-treated mice (TLB stained). Note less annular appearance of large diameter axons with a higher degree of intrinsic parcellation of the demyelinated nerve. (<b>B</b>) Electron micrographs of ON axons from control and treated mice. Along with a large number of myelinated axons (normal) with compact myelin sheaths consisting of several layers, axons covered with a loose myelin envelope of only a few lamellae were regularly encountered in the treated tissue (black arrows). (<b>C</b>) A summary plot of the distribution cross-sectional area of axons with insets highlighting divergence of this parameter for thicker axons (lower inset) and reduced mean cross-sectional area (upper inset) of axons in treated samples. (<b>D</b>) Graphical illustration of the relationship between myelin sheath thickness and axon diameter with regression lines and summary histogram of myelin thickness (inset) show a significant decrease in both parameters in treated mice.</p