Selective blockade of T lymphocyte K(+) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis

Abstract

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4(+) T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype ("chronically activated") with large numbers of Kv1.3 voltage-gated channels (approximately 1500 per cell) and small numbers of IKCa1 Ca(2+)-activated K(+) channels (approximately 50-120 per cell). In contrast, resting T cells displayed 0-10 Kv1.3 and 10-20 IKCa1 channels per cell ("quiescent" phenotype), whereas T cells stimulated once or twice expressed approximately 200 Kv1.3 and approximately 350 IKCa1 channels per cell ("acutely activated" phenotype). Consistent with their channel phenotype, [(3)H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap(22)) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap(22) and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K(+) channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap(22) plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap(22), but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap(22) plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.

Figure 1

Characteristics of the PAS T cell K_V and K_Ca channels. PAS cells were studied 48–72 h after MBP stimulation. (A) Family of K_V currents. The test potential was changed from −60 to +60 mV in 10-mV increments every 30 s (V_1/2 = −33 mV). (B) Use-dependence. We used 200-ms pulses to 40 mV every second. (C and D) Pharmacology. Dose-dependent inhibition of K_V currents by ShK (■, K_d = 9 ± 0.8 pM), ShK-Dap²² (○, K_d = 45 ± 6 pM), MgTX (▴, K_d = 125 ± 12 pM), KTX (□, K_d = 532 ± 72 pM), and ChTX (♦, K_d = 2.5 ± 0.4 nM). Every data point is the mean of three determinations. (E) K_V and IK currents during 200-ms ramp pulses and effect of 1 nM ShK-Dap²² and 200 nM TRAM-34 (Inset, 10 times scale). (F) Pharmacology. Dose-dependent inhibition of IK currents by ChTX (♦, K_d = 5.5 ± 1.1 nM), TRAM-34 (▿, K_d = 19 ± 4 nM), ShK (■, K_d = 25 ± 3 nM) and clotrimazole (⋄, K_d = 91 ± 9 nM).

Figure 2

Four channel-defined rat T cell subsets. Average numbers of Kv1.3 channels per cell (n = 14 to 36 ± SEM) are plotted vs. average numbers of IKCa1 channels (n = 10 to 26). Activated T cells were studied 48–60 h after stimulation. Each cell line/clone or T cell population is numbered as follows. Quiescent group: quiescent normal rat T cells (no. 1). Acutely activated group: normal rat T cells stimulated once (no. 2) or twice with Con A (no. 3), or with PMA (phorbol 12-myristate 13-acetate) + ionomycin (no. 4); MBP-specific CB cells stimulated once with MBP (no. 5). Chronically activated group: T cell lines/clones stimulated ≥8 times by MBP:E10 (no. 6), MBP-33 (no. 7), LR88L1 (no. 8), CB8 (no. 9), CB10 (no. 10), Z (no. 11), PAS (no. 12), ZNP (no. 13); arthritogenic T cell clone (A2b) chronically activated with Mt (no. 14); T cell lines stimulated >8 times by ovalbumin (no. 15) or PPD (no. 16); chronically activated MBP-specific T cells restimulated with Con A:PAS (no. 17) and Z (no. 18). Transition population: T cell lines stimulated 3–6 times with MBP:DAG-S3 (no. 19), Go3-S3 (no. 20), Go1-S3 (no. 21), 71-S5 (no. 22), BP10-S5 (no. 23), Fischer S5 (no. 24), LEG-S6 (no. 25).

Figure 3

Suppression of proliferation by K⁺ channel blockers parallels channel phenotype. (A) Dose-dependent inhibition of [³H]TdR incorporation in MBP-stimulated PAS cells. TRAM-34 (▿, IC₅₀ ≈ 1 μM), ShK (■, IC₅₀ = 30 pM), and ShK-Dap²² (○, IC ₅₀ = 1.4 nM). Proliferation was also inhibited by the Kv1.3 blocker MgTX (IC ₅₀ = 2 nM, not shown). (B) Potentiation of the effects of TRAM-34 on MBP-stimulated PAS cells by combinations with ShK-Dap²²: TRAM-34 alone (▿, IC₅₀ ≈ 1 mM), TRAM-34 + 0.2 nM ShK-Dap²² (▴, IC₅₀ = 10 nM), TRAM-34 + 0.4 nM ShK-Dap²² (○, 1.5 nM), TRAM-34 + 1.6 nM (■). (C) [³H]TdR incorporation into quiescent normal rat T lymphocytes activated for 72 h with Con A in the presence or absence of channel blockers: TRAM-34 (▿, IC₅₀ = 375 nM), ShK (■, IC₅₀ = 50 nM), ShK-Dap²² (○), and MgTX (▴). (D) [³H]TdR incorporation into Con A preactivated normal rat T cells that were restimulated with Con A for a further 72 h: TRAM-34 (▿, IC₅₀ = 200 nM), ShK (■, IC₅₀ = 200 nM), ShK-Dap²² (○), and MgTX (▴). Without stimulus, cpm values were <400 (A and B), <1,000 (C), and <8,000 (D); with stimulus, cpm values ranged from 100 to 250 × 10³ (A and B), 30 × 10³ (C), and 30–50 × 10³ (D).

Figure 4

Blockade of K⁺ channels prevents and treats AT-EAE. Clinical scoring: 0 = no clinical signs, 0.5 = distal limp tail, 1 = limp tail, 2 = mild paraparesis or ataxia, 3 = moderate paraparesis, 4 = complete hind leg paralysis or severe ataxia, 5 = 4 + incontinence, 5.5 = tetraplegia, 6 = death. Mean ± SEM values are shown. (A) Prevention trial. PAS cells were activated in vitro (days −2 to 0) in the presence or absence of channel blockers and washed, and 4 or 5 × 10⁶ viable cells were adoptively transferred i.p. on day 0. Rats were injected with saline (●) or K⁺ channel blockers (▿, TRAM-34; ○, ShK-Dap²²; ▵, ShK-Dap²² + TRAM-34; ■, ShK) from day 0 to day 4 (as shown by the arrows below the x axis). For the control group, data are shown until day 7 when 10 of the 11 animals died; data beyond day 7 for the one surviving animal are not shown. (B and C) Treatment of moderate (B) and severe (C) EAE induced by different batches of PAS cells with varying encephalitogenic potencies. Rats were injected with saline or blockers for 3 days after the onset of clinical signs.