Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum.

In cochlea inner hair cells (IHCs), L-type Ca2؉ channels (LTCCs) formed by ␣1D subunits (D-LTCCs) possess biophysical and pharmacological properties distinct from those of ␣1C containing C-LTCCs. We investigated to which extent these differences are determined by ␣1D itself by analyzing the biophysical and pharmacological properties of cloned human ␣1D splice variants in tsA-201 cells. Variant ␣1D 8A, containing exon 8A sequence in repeat I, yielded ␣1D protein and L-type currents, whereas no intact protein and currents were observed after expression with exon 8B. In whole cell patch-clamp recordings (charge carrier 15-20 mM Ba 2؉ ), ␣1D 8A -mediated currents activated at more negative voltages (activation threshold, ؊45.7 versus ؊31.5 mV, p < 0.05) and more rapidly ( act for maximal inward currents 0.8 versus 2.3 ms; p < 0.05) than currents mediated by rabbit ␣1C. Inactivation during depolarizing pulses was slower than for ␣1C (current inactivation after 5-s depolarizations by 90 versus 99%, p < 0.05) but faster than for LTCCs in IHCs. The sensitivity for the dihydropyridine (DHP) L-type channel blocker isradipine was 8.5-fold lower than for ␣1C. Radioligand binding experiments revealed that this was not due to a lower affinity for the DHP binding pocket, suggesting that differences in the voltage-dependence of DHP block account for decreased sensitivity of D-LTCCs. Our experiments show that ␣1D 8A subunits can form slowly inactivating LTCCs activating at more negative voltages than ␣1C. These properties should allow D-LTCCs to control physiological processes, such as diastolic depolarization in sinoatrial node cells, neurotransmitter release in IHCs and neuronal excitability.L-type Ca 2ϩ channels (LTCCs) 1 form a family of voltagegated Ca 2ϩ channels with high sensitivity to dihydropyridine (DHP) Ca 2ϩ channel modulators. Their Ca 2ϩ -selective pore is formed by different DHP-sensitive ␣1 subunit isoforms (␣1S, ␣1C, ␣1D, ␣1F) together with auxiliary subunits, including ␣2-␦ and ␤ subunits (1-3). Whereas ␣1S and ␣1F expression is restricted to skeletal muscle and the retina, respectively, LTCCs formed by ␣1C (C-LTCCs) and ␣1D (D-LTCCs) subunits are widely expressed in neuronal and (neuro)endocrine cells as well as in electrically excitable cells in the cardiovascular system (4 -9). In most cases, both channel types are even found in the same cells, with D-LTCCs usually being the much less abundant isoform (7).Using D-LTCC-deficient mice, we have previously demonstrated that inward currents through ␣1D form LTCCs with biophysical and pharmacological properties distinct from CLTCCs (4, 6). These include a more negative range of current activation and slower current inactivation during depolarizations, allowing these channels to mediate long lasting Ca 2ϩ influx during weak depolarizations. Such properties allow LTCCs to control tonic neurotransmitter release in hair cells (5, 10), diastolic depolarization in the sinoatrial node (11), and electrical excitability of neurons (12-15).In addition to these different bi...

The Smith-Lemli-Opitz syndrome (SLOS) is an inborn disorder of sterol metabolism with characteristic congenital malformations and dysmorphias. All patients suffer from mental retardation. Here we identify the SLOS gene as a ⌬7-sterol reductase (DHCR7, EC 1.3.1.21) required for the de novo biosynthesis of cholesterol. The human and murine genes were characterized and assigned to syntenic regions on chromosomes 11q13 and 7F5 by f luorescense in situ hybridization. Among the mutations found in patients with the SLOS, are missense (P51S, T93M, L99P, L157P, A247V, V326L, R352W, C380S, R404C, and G410S), nonsense (W151X), and splice site (IVS8-1G>C) mutations as well as an out of frame deletion (720-735 del). The missense mutations L99P, V326L, R352W, R404C, and G410S reduced heterologous protein expression by >90%. Our results strongly suggest that defects in the DHCR7 gene cause the SLOS.

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.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 (ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase (sterol-delta8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-delta8-isomerase (EBP) has been identified and mapped to Xp11.22-p11.23 (refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-delta8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-delta8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive malformation syndrome, ranges in clinical severity from mild dysmorphism and moderate mental retardation to severe congenital malformation and intrauterine lethality. Mutations in the gene for Delta7-sterol reductase (DHCR7), which catalyzes the final step in cholesterol biosynthesis in the endoplasmic reticulum (ER), cause SLOS. We have determined, in 84 patients with clinically and biochemically characterized SLOS (detection rate 96%), the mutational spectrum in the DHCR7 gene. Forty different SLOS mutations, some frequent, were identified. On the basis of mutation type and expression studies in the HEK293-derived cell line tsA-201, we grouped mutations into four classes: nonsense and splice-site mutations resulting in putative null alleles, missense mutations in the transmembrane domains (TM), mutations in the 4th cytoplasmic loop (4L), and mutations in the C-terminal ER domain (CT). All but one of the tested missense mutations reduced protein stability. Concentrations of the cholesterol precursor 7-dehydrocholesterol and clinical severity scores correlated with mutation classes. The mildest clinical phenotypes were associated with TM and CT mutations, and the most severe types were associated with 0 and 4L mutations. Most homozygotes for null alleles had severe SLOS; one patient had a moderate phenotype. Homozygosity for 0 mutations in DHCR7 appears compatible with life, suggesting that cholesterol may be synthesized in the absence of this enzyme or that exogenous sources of cholesterol can be used.

L-type Ca2+ channels are characterized by their unique sensitivity to organic Ca2+ channel modulators like the 1,4-dihydropyridines (DHPs). To identify molecular motifs mediating DHP sensitivity, we transferred this sensitivity from L-type Ca2+ channels to the DHP-insensitive class A brain Ca2+ channel, BI-2. Expression of chimeras revealed minimum sequence stretches conferring DHP sensitivity including segments IIIS5, IIIS6, and the connecting linker, as well as the IVS5-IVS6 linker plus segment IVS6. DHP agonist and antagonist effects are determined by different regions within the repeat IV motif. Sequence regions responsible for DHP sensitivity comprise only 9.4% of the overall primary structure of a DHP-sensitive alpha 1A/alpha 1S construct. This chimera fully exhibits the DHP sensitivity of channels formed by L-type alpha 1 subunits. In addition, it displays the electrophysiological properties of alpha 1A, as well as its sensitivity toward the peptide toxins omega-agatoxin IVA and omega-conotoxin MVIIC.