Isoflurane inhibition of endocytosis is an anesthetic mechanism of action.

The mechanisms of volatile anesthetic action remain among the most perplexing mysteries of medicine. Across phylogeny, volatile anesthetics selectively inhibit mitochondrial complex I, and they also depress presynaptic excitatory signaling. To explore how these effects are linked, we studied isoflurane effects on presynaptic vesicle cycling and ATP levels in hippocampal cultured neurons from wild-type and complex I mutant (Ndufs4(KO)) mice. To bypass complex I, we measured isoflurane effects on anesthetic sensitivity in mice expressing NADH dehydrogenase (NDi1). Endocytosis in physiologic concentrations of glucose was delayed by effective behavioral concentrations of isoflurane in both wild-type (τ [unexposed] 44.8 ± 24.2 s; τ [exposed] 116.1 ± 28.1 s; p < 0.01) and Ndufs4(KO) cultures (τ [unexposed] 67.6 ± 16.0 s; τ [exposed] 128.4 ± 42.9 s; p = 0.028). Increasing glucose, to enhance glycolysis and increase ATP production, led to maintenance of both ATP levels and endocytosis (τ [unexposed] 28.0 ± 14.4; τ [exposed] 38.2 ± 5.7; reducing glucose worsened ATP levels and depressed endocytosis (τ [unexposed] 85.4 ± 69.3; τ [exposed] > 1,000; p < 0.001). The block in recycling occurred at the level of reuptake of synaptic vesicles into the presynaptic cell. Expression of NDi1 in wild-type mice caused behavioral resistance to isoflurane for tail clamp response (EC50 Ndi1(-) 1.27% ± 0.14%; Ndi1(+) 1.55% ± 0.13%) and halothane (EC50 Ndi1(-) 1.20% ± 0.11%; Ndi1(+) 1.46% ± 0.10%); expression of NDi1 in neurons improved hippocampal function, alleviated inhibition of presynaptic recycling, and increased ATP levels during isoflurane exposure. The clear alignment of cell culture data to in vivo phenotypes of both isoflurane-sensitive and -resistant mice indicates that inhibition of mitochondrial complex I is a primary mechanism of action of volatile anesthetics.

Regional knockdown of NDUFS4 implicates a thalamocortical circuit mediating anesthetic sensitivity.

Knockout of the mitochondrial complex I protein, NDUFS4, profoundly increases sensitivity of mice to volatile anesthetics. In mice carrying an Ndufs4lox/lox gene, adeno-associated virus expressing Cre recombinase was injected into regions of the brain postulated to affect sensitivity to volatile anesthetics. These injections generated otherwise phenotypically wild type mice with region-specific, postnatal inactivation of Ndufs4, minimizing developmental effects of gene loss. Sensitivities to the volatile anesthetics isoflurane and halothane were measured using loss of righting reflex (LORR) and movement in response to tail clamp (TC) as endpoints. Knockdown (KD) of Ndufs4 in the vestibular nucleus produced resistance to both anesthetics for movement in response to TC. Ndufs4 loss in the central and dorsal medial thalami and in the parietal association cortex increased anesthetic sensitivity to both TC and LORR. Knockdown of Ndufs4 only in the parietal association cortex produced striking hypersensitivity for both endpoints, and accounted for half the total change seen in the global KO (Ndufs4(KO)). Excitatory synaptic transmission in the parietal association cortex in slices from Ndufs4(KO) animals was hypersensitive to isoflurane compared to control slices. We identified a direct neural circuit between the parietal association cortex and the central thalamus, consistent with a model in which isoflurane sensitivity is mediated by a thalamic signal relayed through excitatory synapses to the parietal association cortex. We postulate that the thalamocortical circuit is crucial for maintenance of consciousness and is disrupted by the inhibitory effects of isoflurane/halothane on mitochondria.

Glutamatergic Neurotransmission Links Sensitivity to Volatile Anesthetics with Mitochondrial Function.

An enigma of modern medicine has persisted for over 150 years. The mechanisms by which volatile anesthetics (VAs) produce their effects (loss of consciousness, analgesia, amnesia, and immobility) remain an unsolved mystery. Many attractive putative molecular targets have failed to produce a significant effect when genetically tested in whole-animal models [1-3]. However, mitochondrial defects increase VA sensitivity in diverse organisms from nematodes to humans [4-6]. Ndufs4 knockout (KO) mice lack a subunit of mitochondrial complex I and are strikingly hypersensitive to VAs yet resistant to the intravenous anesthetic ketamine [7]. The change in VA sensitivity is the largest reported for a mammal. Limiting NDUFS4 loss to a subset of glutamatergic neurons recapitulates the VA hypersensitivity of Ndufs4(KO) mice, while loss in GABAergic or cholinergic neurons does not. Baseline electrophysiologic function of CA1 pyramidal neurons does not differ between Ndufs4(KO) and control mice. Isoflurane concentrations that anesthetize only Ndufs4(KO) mice (0.6%) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) only in Ndufs4(KO) CA1 neurons, while concentrations effective in control mice (1.2%) decreased sEPSC frequencies in both control and Ndufs4(KO) CA1 pyramidal cells. Spontaneous inhibitory postsynaptic currents (sIPSCs) were not differentially affected between genotypes. The effects of isoflurane were similar on evoked field excitatory postsynaptic potentials (fEPSPs) and paired pulse facilitation (PPF) in KO and control hippocampal slices. We propose that CA1 presynaptic excitatory neurotransmission is hypersensitive to isoflurane in Ndufs4(KO) mice due to the inhibition of pre-existing reduced complex I function, reaching a critical reduction that can no longer meet metabolic demands.

Amyloid-beta protein oligomer at low nanomolar concentrations activates microglia and induces microglial neurotoxicity.

Neuroinflammation and associated neuronal dysfunction mediated by activated microglia play an important role in the pathogenesis of Alzheimer disease (AD). Microglia are activated by aggregated forms of amyloid-ß protein (Aß), usually demonstrated in vitro by stimulating microglia with micromolar concentrations of fibrillar Aß, a major component of amyloid plaques in AD brains. Here we report that amyloid-ß oligomer (AßO), at 5-50 nm, induces a unique pattern of microglia activation that requires the activity of the scavenger receptor A and the Ca(2+)-activated potassium channel KCa3.1. AßO treatment induced an activated morphological and biochemical profile of microglia, including activation of p38 MAPK and nuclear factor κB. Interestingly, although increasing nitric oxide (NO) production, AßO did not increase several proinflammatory mediators commonly induced by lipopolyliposaccharides or fibrillar Aß, suggesting that AßO stimulates both common and divergent pathways of microglia activation. AßO at low nanomolar concentrations, although not neurotoxic, induced indirect, microglia-mediated damage to neurons in dissociated cultures and in organotypic hippocampal slices. The indirect neurotoxicity was prevented by (i) doxycycline, an inhibitor of microglia activation; (ii) TRAM-34, a selective KCa3.1 blocker; and (iii) two inhibitors of inducible NO synthase, indicating that KCa3.1 activity and excessive NO release are required for AßO-induced microglial neurotoxicity. Our results suggest that AßO, generally considered a neurotoxin, may more potently cause neuronal damage indirectly by activating microglia in AD.

Potassium channel block by a tripartite complex of two cationophilic ligands and a potassium ion.

Voltage-gated potassium channels (Kv) are targets for drugs of large chemical diversity. Although hydrophobic cations block Kv channels with Hill coefficients of 1, uncharged electron-rich (cationophilic) molecules often display Hill coefficients of 2. The mechanism of the latter block is unknown. Using a combination of computational and experimental approaches, we mapped the receptor for the immunosuppressant PAP-1 (5-(4-phenoxybutoxy)psoralen), a high-affinity blocker of Kv1.3 channels in lymphocytes. Ligand-docking using Monte Carlo minimizations suggested a model in which two cationophilic PAP-1 molecules coordinate a K(+) ion in the pore with their coumarin moieties, whereas the hydrophobic phenoxyalkoxy side chains extend into the intrasubunit interfaces between helices S5 and S6. We tested the model by generating 58 point mutants involving residues in and around the predicted receptor and then determined their biophysical properties and sensitivity to PAP-1 by whole-cell patch-clamp. The model correctly predicted the key PAP-1-sensing residues in the outer helix, the P-loop, and the inner helix and explained the Hill coefficient of 2 by demonstrating that the Kv1.3 pore can accommodate two or even four PAP-1 molecules. The model further explained the voltage-dependence of block by PAP-1 and its thousand-fold selectivity for Kv1.3 over non-Kv1 channels. The 23- to 125-fold selectivity of PAP-1 for Kv1.3 over other Kv1 channels is probably due to its preferential affinity to the C-type inactivated state, in which cessation of K(+) flux stabilizes the tripartite PAP-1:K(+):PAP-1 complex in the pore. Our study provides a new concept for potassium channel block by cationophilic ligands.

Naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a new activator of KCa2 and KCa3.1 potassium channels, potentiates the endothelium-derived hyperpolarizing factor response and lowers blood pressure.

Small-conductance (KCa2.1-2.3) and intermediate-conductance (KCa3.1) calcium-activated K(+) channels are critically involved in modulating calcium-signaling cascades and membrane potential in both excitable and nonexcitable cells. Activators of these channels constitute useful pharmacological tools and potential new drugs for the treatment of ataxia, epilepsy, and hypertension. Here, we used the neuroprotectant riluzole as a template for the design of KCa2/3 channel activators that are potent enough for in vivo studies. Of a library of 41 benzothiazoles, we identified 2 compounds, anthra[2,1-d]thiazol-2-ylamine (SKA-20) and naphtho[1,2-d]thiazol-2-ylamine (SKA-31), which are 10 to 20 times more potent than riluzole and activate KCa2.1 with EC(50) values of 430 nM and 2.9 microM, KCa2.2 with an EC(50) value of 1.9 microM, KCa2.3 with EC(50) values of 1.2 and 2.9 microM, and KCa3.1 with EC(50) values of 115 and 260 nM. Likewise, SKA-20 and SKA-31 activated native KCa2.3 and KCa3.1 channels in murine endothelial cells, and the more "drug-like" SKA-31 (half-life of 12 h) potentiated endothelium-derived hyperpolarizing factor-mediated dilations of carotid arteries from KCa3.1(+/+) mice but not from KCa3.1(-/-) mice. Administration of 10 and 30 mg/kg SKA-31 lowered mean arterial blood pressure by 4 and 6 mm Hg in normotensive mice and by 12 mm Hg in angiotensin-II-induced hypertension. These effects were absent in KCa3.1-deficient mice. In conclusion, with SKA-31, we have designed a new pharmacological tool to define the functional role of the KCa2/3 channel activation in vivo. The blood pressure-lowering effect of SKA-31 suggests KCa3.1 channel activation as a new therapeutic principle for the treatment of hypertension.

Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases.

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2, SAP97, ZIP, p56(lck), and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.