Alterations in membrane caveolae and BKCa channel activity in skin fibroblasts in Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure-function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BK(Ca) K(+) channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BK(Ca) protein but not mRNA expression levels was seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.

Synthesis of an iberiotoxin derivative by chemical ligation: a method for improved yields of cysteine-rich scorpion toxin peptides.

Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val(16) to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp(19) to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited 'native-like' affinity (K(d)=1.9 nM) and specificity towards the BK Ca(2+)-activated K(+) Channel (K(Ca)1.1). This was characterized by the rapid association and slow dissociation rates (k(on)=4.59 x 10(5)M(-1)s(-1); k(off)=8.65 x 10(-4) s(-1)) as determined by inhibition of macroscopic whole-cell currents of cloned human K(Ca)1.1 channel. These results illustrate the successful application of peptide chemical ligation to improve yield of cysteine-rich peptide toxins over traditional solid phase peptide synthesis. Native chemical ligation is a promising method for improving production of biologically active disulfide containing peptide toxins, which have diverse applications in studies of ion-channel function.

The dipeptidyl-peptidase-like protein DPP6 determines the unitary conductance of neuronal Kv4.2 channels.

The neuronal subthreshold-operating A-type K(+) current regulates electrical excitability, spike timing, and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (gamma) of neuronal somatodendritic A-type K(+) channels composed of Kv4 pore-forming subunits is larger (approximately 7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (approximately 4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like protein-6 DPP6-S to the gamma of native [cerebellar granule neuron (CGN)] and reconstituted Kv4.2 channels. Coexpression of Kv4.2 proteins with DPP6-S was sufficient to match the gamma of native CGN channels; and CGN Kv4 channels from dpp6 knock-out mice yielded a gamma indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in gamma. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the dpp6 gene.

The neuronal Kv4 channel complex.

Kv4 channel complexes mediate the neuronal somatodendritic A-type K(+) current (I(SA)), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated alpha-subunits (Shal/Kv4) and at least two classes of auxiliary beta-subunits: KChIPs (K(+)-Channel-Interacting-Proteins) and DPLPs (Dipeptidyl-Peptidase-Like-Proteins). Here, we review our investigations of Kv4 gating mechanisms and functional remodeling by specific auxiliary beta-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn(2+) site in the T1 domain undergoes a conformational change tightly coupled to voltage-dependent gating and is targeted by nitrosative modulation; and (3) discrete and specific interactions mediate the effects of KChIPs and DPLPs on activation, inactivation and permeation of Kv4 channels. These studies are shedding new light on the molecular bases of I(SA) function and regulation.

Effect of agents modifying the membrane dipole potential on properties of syringomycin E channels.

We evaluated the effect of agents modifying the membrane dipole potential: phloretin, 6-ketocholestanol and RH 421 on the properties of single channels formed by lipodepsipeptide syringomycin E (SRE) in planar lipid bilayers. SRE forms two conductive states in lipid bilayers: "small" and "large." Large SRE channels are clusters of several small ones, demonstrating synchronous openings and closures. The increase in the membrane dipole potential led to (i) an increase in SRE channel conductance, (ii) an increase in the channel's lifetime, and (iii) a decrease in a number of synchronously operating small channels in the clusters. Overall, the results support the model of the small SRE channel synchronization in the cluster as voltage-dependent orientation of the lipid dipoles associated with the channel pores.

Sphingolipids influence the sensitivity of lipid bilayers to fungicide, syringomycin E.

Sphingolipids with long chain bases hydroxylated at the C4 position are a requisite for the yeast, Saccharomyces cerevisia, to be sensitive to the ion channel forming antifungal agent, syringomycin E (SRE). A mutant S. cerevisiae strain, Deltasyr2, having sphingolipids with a sphingoid base devoid of C4-hydroxylation, is resistant to SRE. To explore the mechanism of this resistance, we investigated the channel forming activity of SRE in lipid bilayers of varying composition. We found that the addition of sphingolipid-rich fraction from Deltasyr2 to the membrane-forming solution (DOPS/DOPE/ergosterol) resulted in lipid bilayers with lower sensitivity to SRE compared with those containing sphingolipid fraction from wild-type S. cerevisiae. Other conditions being equal, the rate of increase of bilayer conductance was about 40 times slower, and the number of SRE channels was about 40 times less, with membranes containing Deltasyr2 versus wild-type sphingolipids. Deltasyr2 sphingolipids altered neither SRE single channel conductance nor the gating charge but the ability of SRE channels to open synchronously was diminished. The results suggest that the resistance of the Deltasyr2 mutant to SRE may be partly due to the ability of sphingolipids without the C4 hydroxyl group to decrease the channel forming activity of SRE.

Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus.

BACKGROUND: The channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers. RESULTS: Both PHA-E and RCA-I almost exclusively labeled an 82-84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82-84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82-84 kDa which may be interpreted as a component of a multimeric receptor/channel complex. CONCLUSIONS: The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata.