Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca2+ influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

Voltage-gated Na(+) currents play critical roles in shaping electrogenesis in neurons. Here, we have identified a TTX-resistant Na(+) current (TTX-R I(Na)) in duodenum myenteric neurons of guinea pig and rat and have sought evidence regarding the molecular identity of the channel producing this current from the expression of Na(+) channel alpha subunits and the biophysical and pharmacological properties of TTX-R I(Na). Whole-cell patch-clamp recording from in situ neurons revealed the presence of a voltage-gated Na(+) current that was highly resistant to TTX (IC(50), approximately 200 microm) and selectively distributed in myenteric sensory neurons but not in interneurons and motor neurons. TTX-R I(Na) activated slowly in response to depolarization and exhibited a threshold for activation at -50 mV. V(1/2) values of activation and steady-state inactivation were -32 and -31 mV in the absence of fluoride, respectively, which, as predicted from the window current, generated persistent currents. TTX-R I(Na) also had prominent ultraslow inactivation, which turns off 50% of the conductance at rest (-60 mV). Substituting CsF for CsCl in the intracellular solution shifted the voltage-dependent parameters of TTX-R I(Na) leftward by approximately 20 mV. Under these conditions, TTX-R I(Na) had voltage-dependent properties similar to those reported previously for NaN/Na(V)1.9 in dorsal root ganglion neurons. Consistent with this, reverse transcription-PCR, single-cell profiling, and immunostaining experiments indicated that Na(V)1.9 transcripts and subunits, but not Na(V)1.8, were expressed in the enteric nervous system and restricted to myenteric sensory neurons. TTX-R I(Na) may play an important role in regulating subthreshold electrogenesis and boosting synaptic stimuli, thereby conferring distinct integrative properties to myenteric sensory neurons.

The aim of the present paper was to determine the electrotonic properties of myenteric neurones, using patchclamp recording from non-dissociated myenteric neurones, with emphasis on the identification and quantitation of the ionic currents that modulate the resting membrane potential of AH neurones. Recordings were made with a technique we have recently developed for patch-clamp recording from intact ganglia (Kunze et al. 2000).The electrophysiological properties of myenteric neurones in intact ganglia from the small intestine of the guinea-pig have been investigated previously by intracellular recordings in myenteric plexus/longitudinal muscle preparations. The first intracellular recording studies were performed using duodenal (Hirst et al. 1974) and ileal tissue (Nishi & North, 1973). In both of these parts of the intestine, the studies separated the myenteric neurones into two groups, S and AH neurones, terms that were introduced in 1974 for duodenal neurones (Hirst et al. 1974). S neurones were so named because they received prominent fast synaptic inputs, while AH neurones did not. AH neurones were so named because the action potential is followed by a long-lasting afterhyperpolarization (AHP). It was later demonstrated that both cell types receive slow EPSPs (Wood & Meyer, 1978; Johnson et al. 1980 Johnson et al. , 1981 Bornstein et al. 1984). Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact gangliaFrançois Rugiero, Maurice Gola, Wolf A. A. Kunze *, Jean-Claude Reynaud, John B. Furness * and Nadine Clerc Laboratoire 'Intégration des Informations Sensorielles' (ITIS), CNRS, Bâtiment LNB, No. 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France and *Department of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC 3010, Australia Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of _47 ± 6 mV and input resistances (R in ) of 713 ± 49 MV at voltages ranging from _90 to _40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inwardrectifier current (I Kir ) decreased R in to 103 ± 10 MV. AH neurones had resting potentials of _57 ± 4 mV and R in was 502 ± 27 MV. R in fell to 194 ± 16 MV upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, I h , and to I Kir . Resting potential and R in exhibited a low sensitivity to changes in [K + ] o in both AH and S neurones. This indicates that both cells have a low background K + permeability. The cationic current, I h , contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of _72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e-fold change. I h has relatively fast, voltage-dependent kinetics, with on and off time constants in the range ...

Intrinsic primary afferent neurons in the small intestine are exposed to distortion of their processes and of their cell bodies. Recordings of mechanosensitivity have previously been made from these neurons using intracellular microelectrodes, but this form of recording has not permitted detection of generator potentials from the processes, or of responses to cell body distortion. We have developed a technique to record from enteric neurons in situ using patch electrodes. The mechanical stability of the patch recordings has allowed recording in cell‐attached and whole cell configuration during imposed movement of the neurons. Pressing with a fine probe initiated generator potentials (14 ± 9 mV) from circumscribed regions of the neuron processes within the same myenteric ganglion, at distances from 100 to 500 μm from the cell body that was patched. Generator potentials persisted when synaptic transmission was blocked with high Mg2+, low Ca2+ solution. Soma distortion, by pressing down with the whole cell recording electrode, inhibited action potential firing. Consistent with this, moderate intra‐electrode pressure (10 mbar; 1 kPa) increased the opening probability of large‐conductance (BK) potassium channels, recorded in cell‐attached mode, but suction was not effective. In outside‐out patches, suction, but not pressure, increased channel opening probability. Mechanosensitive BK channels have not been identified on other neurons. The BK channels had conductances of 195 ± 25 pS. Open probability was increased by depolarization, with a half‐maximum activation at a patch potential of 20 mV and a slope factor of 10 mV. Channel activity was blocked by charybdotoxin (20 nM). Stretch that increased membrane area under the electrode by 15 % was sufficient to double open probability. Similar changes in membrane area occur when the intestine changes diameter and wall tension under physiological conditions. Thus, the intestinal intrinsic primary afferent neurons are detectors of neurite distortion and of compression of the soma, these stimuli having opposite effects on neuron excitability.

The ATP binding cassette transporter ABC1 is a 220-kDa glycoprotein expressed by macrophages and required for engulfment of cells undergoing programmed cell death. Since members of this family of proteins such as P-glycoprotein and cystic fibrosis transmembrane conductance regulator share the ability to transport anions, we have investigated the transport capability of ABC1 expressed in Xenopus oocytes using iodide efflux and voltage-clamp techniques. We report here that ABC1 generates an anion flux sensitive to glibenclamide, sulfobromophthalein, and blockers of anion transporters. The anion flux generated by ABC1 is upregulated by orthovanadate, cAMP, protein kinase A, and okadaic acid. In other ABC transporters, mutating the conserved lysine in the nucleotide binding folds was found to severely reduce or abolish hydrolysis of ATP, which in turn altered the activity of the transporter. In ABC1, replacement of the conserved lysine 1892 in the Walker A motif of the second nucleotide binding fold increased the basal ionic flux, did not alter the pharmacological inhibitory profile, but abolished the response to orthovanadate and cAMP agonists. Therefore, we conclude that ABC1 is a cAMP-dependent and sulfonylureasensitive anion transporter.