Kv1.3 Channel Up-Regulation in Peripheral Blood T Lymphocytes of Patients With Multiple Sclerosis

Figures

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  FIGURE 1. Patch clamp data from T cells of MS patients and healthy controls. (A): Higher current responses in MS T cells. Whole-cell currents from T lymphocytes bathed in extracellular solution of pH 7.4 with their membrane clamped at −90 mV. MS T cells (N = 855) have higher peak KV1.3 amplitudes than controls (N = 1,258). ( A1–A3): Representative KV1.3 currents elicited by 1000-ms depolarizing pulses in 15-mV increments, given every 60 s are displayed at physiologically relevant potentials (−50 to +10 mV) for reasons of clarity. (B): T cell Kv1.3 currents in SPMS have significantly higher mean peak amplitudes than both RRMS and healthy controls. (B1): Increased peak Kv1.3 conductance, in MS patients compared to healthy individuals. Peak current amplitudes in response to voltage step protocols were converted to conductance (g) values, averaged and plotted against the corresponding voltage for SPMS (* ), RRMS (○ ) and healthy controls (□ ) (g-V curves). The average maximal peak conductance was 5.49 ± 0.22 for SPMS (n = 18 patients, N = 415 cells), 4.7 ± 0.19 for RRMS (n = 20 patients, N = 440 cells) and 372 ± 0.25 for healthy individuals (n = 21, N = 542 cells) (p < 0.001). Data shown are…

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  FIGURE 2. Specific electrophysiological protocols exclude TASK-2 contribution to the increased potassium conductance of MS T lymphocytes. (A): T cells from either MS patients or healthy controls present similar current-responses in alkaline solution at a 0 mV holding potential: Whole-cell currents recorded from T lymphocytes bathed in an alkaline (pH = 8.5) extracellular solution with their membranes clamped at 0 mV. ( A1–A3): Whole-cell current responses to 15 mV voltage-steps, given every 5 s from T lymphocytes of healthy controls, RRMS and SPMS patients. ( A4): Average peak currents (at +40 mV) did not differ significantly between groups. Average values ±SE were: 11.9 ± 0,48 pA for SPMS (n = 12, N = 235 cells), 12.2 ± 0,53 pA for RRMS (n = 14, N = 282 cells) and 11.2 pA ± 0,6 for healthy subjects (n = 15, N = 310 cells) (ns: non-significant). Method: One way ANOVA with Bonferroni post hoc correction. (B). T cells of MS patients exhibit higher K+ currents than controls in acidic extracellular solution: Whole-cell currents recorded from T cells bathed in acidic (pH = 6.3) extracellular medium with their membrane held at −90 mV. KV1.3 currents from T cells of healthy controls, RRMS and…

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  FIGURE 3. (A): Calculated KV1.3 channel densities per T lymphocyte from whole-cell currents recorded from a holding potential of −90 mV. (A1): KV1.3 channel densities in MS patients (n = 38, N = 852 cells) are significantly higher (p < 0.001), compared to healthy control subjects (n = 21, N = 540 cells), patients with: clinically isolated Syndrome (CIS) (n = 6, N = 130 cells), other neurological disorders (OND) (n = 16 patients, N = 335 cells), and other inflammatory neurological disorders (OIND) (n = 9 patients, N = 253 cells). Each symbol represents mean values for each individual patient and error bars are standard deviation of the mean. Red horizontal lines with red error bars correspond to the average value ±standard error for each group. Comparisons are shown only for statistically significant differences (٭٭٭: p < 0.001). One way ANOVA with Bonferroni post hoc correction was applied for statistical analysis. ( A2): Sub-grouping of MS patients showed significantly higher KV1.3 channel densities in Secondary Progressive Multiple Sclerosis (SPMS) (n = 18 patients, N = 415 cells), compared to Relapse Remitting Multiple Sclerosis (RRMS) (n = 20 patients. N = 440 cells). Values shown are…

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  FIGURE 4. (A). Expression of Kv1.3, TASK-2 and KCa3.1 mRNA in T lymphocytes of patients with multiple sclerosis. (A1): Agarose gel image from PCR products of KV1.3, TASK-2, KCa3.1 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in T lymphocytes of four healthy control subjects (lanes CNT 1–4) and four patients with multiple sclerosis (lanes MS 1–4). The PCR products demonstrated: a band with a molecular size of 103 bp, corresponding to KV1.3, a band of 106 bp corresponding to TASK-2, a band of 125 bp corresponding to KCa3.1, and a band of 117 bp corresponding to GAPDH transcripts. MS patients gave KV1.3 bands of higher intensity (1.32 ± 0.18, n = 34) compared to healthy controls (0.76 ± 0.16, n = 20, p < 0.001) and similar intensities for the TASK-2 (controls 0.78 ± 0.12, MS 0.77 ± 0.1) and KCa3.1 (control 1.12 ± 0.14, MS 1.06 ± 0.11) bands in these samples. In the same samples the bands of the housekeeping gene GAPDH had similar intensities. ( A2): Agarose gel image of the PCR products of GAPDH, Kv1.3, TASK-2 and KCa3.1 amplified from cDNA derived from commercially available human whole brain (HWB) RNA. HWB was used as positive internal control for all the sets…

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  FIGURE 5. Kv1.3 channel up-regulation enhances signaling in MS T cells. Upon TCR antigenic (Ag) stimulation, the IP3-induced calcium release from the intracellular stores (ER) causes on one hand membrane depolarization (Vm), followed by a Kv1.3 channel-mediated hyperpolarization and on the other, the formation of conductive Calcium Release Activated Calcium (CRAC) channels; in MS-T cells this hyperpolarization will be of increased magnitude due to higher Kv1.3 channel density. This in turn will translate into a stronger than physiological driving force for calcium ions (Ca2+) through CRAC channels and therefore a cytosolic calcium signal of amplified intensity and duration that in turn will potentiate the effectiveness of the immune stimulus, and may even allow sub-threshold stimuli to reach activation threshold, priming T cell activation that otherwise would not. Additionally all calcium-dependent processes that follow the successful activation of MS T cells, such as cytokine production, cytokine secretion and proliferation, will be heightened. Moreover, the immune stimulus-induced Kv1.3 activity in these cells, will increase β1-integrin activation, facilitating their adhesion and…