T-type calcium channels are widely distributed in diverse tissues and dysfunctions of these channels contribute to a variety of disorders and diseases. Notably, few specific ligands are available for physiological identification of T-type calcium channels. Here we identify u-agatoxin IIA (u-Aga-IIA), a polypeptide toxin purified from venom of American Funnel Web spider, Agelenopsis aperta, as a high affinity low voltage-activated (T-type) calcium channel antagonist. In whole cell recordings of the human a 1I channel stably expressed in HEK cells, u-Aga-IIA partially inhibited T-type current with an EC 50 of 1.05 5 0.62 nM. u-Aga-IIA also partially blocked a 1B calcium channels with a higher efficacy than its effect on a 1I channel, with a comparable EC 50 of 0.1750.056 nM. u-Aga-IIA partially inhibited T-type and N-type calcium current at saturating concentrations without shifting the I-V curve. We also developed a heterologous expression system (E. coli) and a modified on-column protein refolding method for production of a u-Aga-IIA isoform, u-Agatoxin IIC (u-Aga-IIC). Recombinant u-Aga-IIC exhibited similar RP-HPLC elution profiles as u-Aga-IIA and blocked a 1I/ a 1B channels with high potency (EC 50 of 1.01 5 0.38 and 0.1650.049, respectively). The high affinities of u-Aga-IIA and u-Aga-IIC for a 1I and a 1B calcium channels indicates the presence of an evolutionarily conserved binding site on high-and low voltage-activated calcium channels. With the successfully production and refolding of recombinant u-Aga-IIC, a valuable tool has become available for further studies of calcium channel pharmacology and function. 2873-Plat Caveolin-3 Inhibits Ca v 3.2 (a1H) Currents and Regulates Hypertrophic Signaling in Ventricular Myocytes

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by CaV1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3′,5′-monophosphate–dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac CaV1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of CaV1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α2δ1 and β2b subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of CaV1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal CaV1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for CaV1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional signi¢cance in Aspergillus species is unknown. We here identi¢ed and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3P P,4P P-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a signi¢cantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.

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