Tissue expression and distribution of the high-conductance Ca(2+)-activated K+ channel Slo was investigated in rat brain by immunocytochemistry, in situ hybridization, and radioligand binding using the novel high-affinity (Kd 22 pM) ligand [3H]iberiotoxin-D19C ([3H]IbTX-D19C), which is an analog of the selective maxi-K peptidyl blocker IbTX. A sequence-directed antibody directed against Slo revealed the expression of a 125 kDa polypeptide in rat brain by Western blotting and precipitated the specifically bound [3H]IbTX-D19C in solubilized brain membranes. Slo immunoreactivity was highly concentrated in terminal areas of prominent fiber tracts: the substantia nigra pars reticulata, globus pallidus, olfactory system, interpeduncular nucleus, hippocampal formation including mossy fibers and perforant path terminals, medial forebrain bundle and pyramidal tract, as well as cerebellar Purkinje cells. In situ hybridization indicated high levels of Slo mRNA in the neocortex, olfactory system, habenula, striatum, granule and pyramidal cell layer of the hippocampus, and Purkinje cells. The distribution of Slo protein was confirmed in microdissected brain areas by Western blotting and radioligand-binding studies. The latter studies also established the pharmacological profile of neuronal Slo channels. The expression pattern of Slo is consistent with its targeting into a presynaptic compartment, which implies an important role in neural transmission.

cross-presentation ͉ dendritic cells ͉ epidermis ͉ mouse or murine ͉ epicutaneous

No abstract

We have investigated the functional consequences of three P/Q-type Ca 2؉ channel ␣1A (Ca v 2.1␣ 1 ) subunit mutations associated with different forms of ataxia (episodic ataxia type 2 (EA-2), R1279Stop, AY1593/1594D; progressive ataxia (PA), G293R). Mutations were introduced into human ␣1A cDNA and heterologously expressed in Xenopus oocytes or tsA-201 cells (with ␣ 2 ␦ and ␤1a) for electrophysiological and biochemical analysis. G293R reduced current density in both expression systems without changing single channel conductance. R1279Stop and AY1593/1594D protein were expressed in tsA-201 cells but failed to yield inward barium currents (I Ba ). However, AY1593/1594D mediated I Ba when expressed in oocytes. G293R and AY1593/1594D shifted the current-voltage relationship to more positive potentials and enhanced inactivation during depolarizing pulses (3 s) and pulse trains (100 ms, 1 Hz). Mutation AY1593/ 1594D also slowed recovery from inactivation. Single channel recordings revealed a change in fast channel gating for G293R evident as a decrease in the mean open time. Our data support the hypothesis that a pronounced loss of P/Q-type Ca 2؉ channel function underlies the pathophysiology of EA-2 and PA. In contrast to other EA-2 mutations, AY1593/1594D and G293R form at least partially functional channels.Genetic defects within the pore-forming Ca v 2.1␣ 1 (␣1A) subunit of neuronal voltage-gated P/Q-type Ca 2ϩ channels are associated with inherited human neurological diseases, such as Familial Hemiplegic Migraine (FHM), 1 Episodic Ataxia Type 2 (EA-2), progressive cerebellar ataxia, and epilepsy (1-6). Therefore, neuronal Ca 2ϩ channel dysfunction represents an important pathophysiological mechanism that may also underlie more common forms of migraine, epilepsy, and neurodegenerative processes.By mediating depolarization-induced Ca 2ϩ influx into dendrites, cell bodies, and nerve terminals, neuronal voltage-gated Ca 2ϩ channels control important neuronal processes. This includes fast neurotransmitter release, gene expression, neuronal plasticity, migration, and differentiation (7). P/Q-type Ca 2ϩ channels are very tightly coupled to neurotransmitter release in many neurons (see e.g. Refs. 8 and 9) and mediate most of the depolarization-induced Ca 2ϩ current in cerebellar Purkinje cells (10). They exist as hetero-oligomeric complexes of ␣1A subunits together with accessory subunits (especially ␣2␦ and ␤ (11)). ␣1A subunit-mediated neurotransmitter release is tightly controlled by other neurotransmitters (e.g. via protein kinase C phosphorylation and G-protein ␤␥ subunits (12-14)) and by their direct association with synaptic vesicles through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (15). Hence, these channels are ideally suited to fine-tune synaptic strength. This also explains why genetic defects, despite causing only minor changes in channel gating or expression, can lead to the above-mentioned clinical symptoms in humans and to the severe neurological abnormalities ...

Monoiodotyrosine margatoxin ([125I]MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This Kd value was confirmed by kinetic experiments (Kd of 0.07 pM), competition assays employing native margatoxin (MgTX) (Ki of 0.15 pM), and receptor saturation studies (Kd of 0.18 pM). Thus, this toxin represents the highest affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date. [125I]MgTX binding in this system is modulated by charybdotoxin (Ki of 5 pM), kaliotoxin (Ki of 1.5 pM), and the agitoxins I and II (Ki's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner. Moreover, alpha-dendrotoxin displayed a Ki value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex. [125I]MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased. Stimulation of binding results from an allosteric interaction that decreases Kd, whereas inhibition is due to an ionic strength effect. Affinity labeling of the binding site in rat brain synaptic plasma membranes employing [125I]MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74,000. Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63,000.(ABSTRACT TRUNCATED AT 250 WORDS)