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H Glossmann

Publications and source records attributed to H Glossmann.

At least 19 recordsLinked to original sources
PubMed2001

alpha 1D (Cav1.3) subunits can form l-type Ca2+ channels activating at negative voltages.

In cochlea inner hair cells (IHCs), L-type Ca(2+) channels (LTCCs) formed by alpha1D subunits (D-LTCCs) possess biophysical and pharmacological properties distinct from those of alpha1C containing C-LTCCs. We investigated to which extent these differences are determined by alpha1D itself by analyzing the biophysical and pharmacological properties of cloned human alpha1D splice variants in tsA-201 cells. Variant alpha1D(8A,) containing exon 8A sequence in repeat I, yielded alpha1D 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+)), alpha1D(8A) - mediated currents activated at more negative voltages (activation threshold, -45.7 versus -31.5 mV, p < 0.05) and more rapidly (tau(act) for maximal inward currents 0.8 versus 2.3 ms; p < 0.05) than currents mediated by rabbit alpha1C. Inactivation during depolarizing pulses was slower than for alpha1C (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 alpha1C. 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 alpha1D(8A) subunits can form slowly inactivating LTCCs activating at more negative voltages than alpha1C. 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.

Amino Acid Sequence↗
PubMed2001

Mechanism of dihydropyridine interaction with critical binding residues of L-type Ca2+ channel alpha 1 subunits.

We investigated the mechanism of interaction of individual L-type channel amino acid residues with dihydropyridines within a dihydropyridine-sensitive alpha1A subunit (alpha1A(DHP)). Mutation of individual residues in repeat III and expression in Xenopus oocytes revealed that Thr(1393) is not required for dihydropyridine interaction but that bulky side chains (tyrosine, phenylalanine) in this position sterically inhibit dihydropyridine coordination. In position 1397 a side chain carbonyl group was required for high antagonist sensitivity. Agonist function required the complete amide group of a glutamine residue. Val(1516) and Met(1512) side chains were required for agonist (Val(1516)) and antagonist (Val(1516), Met(1512)) sensitivity. Replacement of Ile(1504) and Ile(1507) by alpha1A phenylalanines was tolerated. Substitution of Thr(1393) by phenylalanine or Val(1516) by alanine introduced voltage dependence of antagonist action into alpha1A(DHP), suggesting that these residues form part of a mechanism mediating voltage dependence of dihydropyridine sensitivity. Our data provide important insight into dihydropyridine binding to alpha1A(DHP) which could facilitate the development of alpha1A-selective modulators. By modulating P/Q-type Ca(2+) channels such drugs could serve as new anti-migraine therapeutics.

Alanine↗
PubMed2001

7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome.

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7(-/-) mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.

Animals↗
PubMed2000

Conserved Ca2+-antagonist-binding properties and putative folding structure of a recombinant high-affinity dihydropyridine-binding domain.

Sensitivity to 1,4-dihydropyridines (DHPs) can be transferred from L-type (alpha1C) to non-L-type (alpha1A) Ca(2+) channel alpha1 subunits by the mutation of nine pore-associated non-conserved amino acid residues, yielding mutant alpha1A(DHP). To determine whether the hallmarks of reversible DHP binding to L-type Ca(2+) channels (nanomolar dissociation constants, stereoselectivity and modulation by other chemical classes of Ca(2+) antagonist drugs) were maintained in alpha1A(DHP), we analysed the pharmacological properties of (+)-[(3)H]isradipine-labelled alpha1A(DHP) Ca(2+) channels after heterologous expression. Binding of (+)-isradipine (K(i) 7.4 nM) and the non-benzoxadiazole DHPs nifedipine (K(i) 86 nM), (+/-)-nitrendipine (K(i) 33 nM) and (+/-)-nimodipine (K(i) 67 nM) to alpha1A(DHP) occurred at low nanomolar K(i) values. DHP binding was highly stereoselective [25-fold higher affinity for (+)-isradipine]. As with native channels it was stimulated by (+)-cis-diltiazem, (+)-tetrandrine and mibefradil. This suggested that the three-dimensional architecture of the channel pore was maintained within the non-L-type alpha1A subunit. To predict the three-dimensional arrangement of the DHP-binding residues we exploited the X-ray structure of a recently crystallized bacterial K(+) channel (KcsA) as a template. Our model is based on the assumption that the Ca(2+) channel S5 and S6 segments closely resemble the KcsA transmembrane folding architecture. In the absence of three-dimensional structural data for the alpha1 subunit this is currently the most reasonable approach for modelling this drug-interaction domain. Our model predicts that the previously identified DHP-binding residues form a binding pocket large enough to co-ordinate a single DHP molecule. It also implies that the four homologous Ca(2+) channel repeats are arranged in a clockwise manner.

Amino Acid Sequence↗
PubMed2000

Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics.

Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q-type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.

Animals↗
PubMed2000

Expression of the purported sigma(1) (sigma(1)) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy.

Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of sigma receptor binding sites (sigma(1) and sigma(2)) is now well established. The recent cloning of the mouse, guinea pig and human sigma(1) receptors has allowed the study of the discrete distribution of the sigma(1) receptor mRNA in rodent and human brain tissues using in situ hybridization. Overall, the sites of expression of specific sigma(1) receptor mRNA signals were in accordance to the anatomical distribution of sigma(1) receptor protein first established by quantitative receptor autoradiography. Specific sigma(1) receptor hybridization signals were found to be widely, but discretely distributed, in mouse and guinea pig brain tissues. The highest levels of transcripts were seen in various cranial nerve nuclei. Lower, but still high hybridization signals were observed in mesencephalic structures such as the red nucleus, periaqueductal gray matter and substantia nigra, as well as in some diencephalic structures including such as the habenula and the arcuate, paraventricular and ventromedial hypothalamic nuclei. Superficial (I-II) and deeper (IV-VI) cortical laminae were moderately labeled in the mouse brain. Moderate levels of sigma(1) receptor mRNA were also found in the pyramidal cell layer and the dentate gyrus of the hippocampal formation. Other structures such as the thalamus and amygdaloid body also expressed the sigma(1) receptor mRNA although to a lesser extent. In murine peripheral tissues, strong hybridization signals were observed in the liver, white pulp of the spleen and the adrenal gland. In the postmortem human brain, moderate levels of sigma(1) receptor mRNA, distributed in a laminar fashion, were detected in the temporal cortex with the deeper laminae (IV-VI) being particularly enriched. In the hippocampal formation, the strongest hybridization signals were observed in the dentate gyrus while all other subfields of the human hippocampal formation expressed lower levels of the sigma(1) receptor mRNA. Antisense oligodeoxynucleotides against the purported sigma(1) receptor were used next to investigate the possible role of this receptor in dizocilpine (MK-801)/NMDA receptor blockade-induced amnesia. Following a continuous intracerebroventricular infusion of a specific sigma(1) receptor antisense into the third ventricle (0.4 nmol/h for 5 days), sigma(1)/[3H](+)pentazocine binding was significantly reduced in mouse brain membrane homogenates while a scrambled antisense control was without effect. Moreover, the sigma(1) receptor antisense treatments (5 nmol/injection, every 12 hx3 or 0.4 nmol/h for 5 days) attenuated (+)MK-801/NMDA receptor blockade-induced cognitive deficits in the treated mice while a scrambled antisense control had no effect. Taken together, these results demonstrate the widespread, but discrete, distribution of the sigma(1) receptor mRNA in the mammalian central nervous system. Moreover, antisense treatments against the purported sigma(1) receptor gene reduced specific sigma(1)/[3H](+)pentazocine binding and modulated cognitive behaviors associated with NMDA receptor blockade providing further evidence for the functional relevance of the cloned gene.

Amnesia↗
PubMed2000

Genetic defects in postsqualene cholesterol biosynthesis.

In humans and mice, four different genetic defects in the nine biosynthetic steps from lanosterol to cholesterol have been identified. They impair the activity of a putative C3-sterol dehydrogenase (Nshdl, X-linked dominant bare patches/striated mutation in mice), the sterol delta 8-delta 7 isomerase/EBP (Ebp, X-linked dominant tattered mutation in mice; chondrodysplasia punctata (CDPX2) in humans), the delta 24-sterol reductase (autosomal recessive desmosterolosis) and the delta 7-sterol reductase (DHCR7 gene, autosomal recessive Smith-Lemli-Opitz syndrome in humans). These inborn errors in postsqualene cholesterol metabolism result in dysmorphogenetic syndromes of variable severity. The X-linked dominant mutations result in mosaicism in females, as a result of X-inactivation, and midgestational lethality in males. The mechanisms by which the depletion of cholesterol or the accumulation of intermediates impair morphogenetic programs are unclear. So far, no cellular processes that require an intact cholesterol biosynthetic pathway have been identified, although the morphogenetic hedgehog-patched signaling cascade is a candidate.

Animals↗
PubMed2000

Mutational spectrum in the Delta7-sterol reductase gene and genotype-phenotype correlation in 84 patients with Smith-Lemli-Opitz syndrome.

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.

Adolescent↗
PubMed1999

WIN 17317-3, a new high-affinity probe for voltage-gated sodium channels.

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.

Animals↗
PubMed1999

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor.

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.

Amino Acid Sequence↗
PubMed1999

Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization.

The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Delta8-Delta7 isomerization-deficient erg2-3 yeast strain. Ala scanning mutagenesis was used to identify residues in the four putative transmembrane alpha-helices of hEBP that are required for catalytic activity. Isomerization was assayed in vivo by spectrophotometric quantification of Delta5,7-sterols. Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A-, and W197A-expressing yeast strains contained 10% or less of wild-type (wt) Delta5,7-sterols. All substitutions of these six residues with functionally or structurally similar amino acid residues failed to fully restore catalytic activity. Mutants E81D, T126S, N194Q, and W197F, but not H77N and E123D, still bound the enzyme inhibitor 3H-ifenprodil. Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding [Moebius et al. (1996) Biochemistry 35, 16871-16878]. His77, Glu81, Glu123, Thr126, Asn194, and Trp197 are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are proposed to line the catalytic cleft. Ala mutants of Trp102, Tyr105, Asp109, Arg111, and Tyr112 in a conserved cytoplasmic domain (WKEYXKGDSRY) between transmembrane segments 2 and 3 contained less than 10% of wt Delta5,7-sterols, implying that this region also could be functionally important. The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional consequences of mutations in sterol modifying enzymes.

Alanine↗
PubMed1999

Molecular mechanisms of the effects of sildenafil (VIAGRA).

The molecular mechanisms of the effects of sildenafil, a specific inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterases are briefly reviewed. The second messenger cGMP as well as its molecular targets (with the exception of the photoreceptor signal transduction machinery) have long played an underdog role compared with cyclic adenosine monophosphate and other signalling molecules such as inositoltrisphosphate. The same holds for guanylyl cyclase, which, albeit being the main effector molecule of the gaseous neurotransmitters carbon monoxide and nitric oxide (NO), has received much less attention relative to its activators and their synthases. Stimulation of the arginine --> NO --> cGMP pathway by bypassing NO-synthase is a well-established pharmacological principle in the treatment of cardiovascular disorders. In contrast, local application of NO-donors or oral feeding of excessive amounts of precursor amino acid L-arginine to treat erectile dysfunction were met with variable success or failure. The advent of a new principle, amplification of the NO-signaling cascade by means of target organ selective phosphodiesterase inhibition, has renewed interest in phosphodiesterases and cGMP.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hünermann syndrome.

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.

Adolescent↗
PubMed1999

Molecular genetics of the Smith-Lemli-Opitz syndrome and postsqualene sterol metabolism.

The Smith-Lemli-Opitz syndrome is a disorder of morphogenesis resulting from an enzymatic defect in the last step of cholesterol metabolism (reduction of 7-dehydrocholesterol). Analysis of the defective gene and identification of mutations therein have paved the way for the study of the molecular genetics of the disorder which is caused by numerous different mutations. Future efforts should identify a postulated intracellular signalling activity of sterol intermediates, isolate proteins that govern the sterol traffic between intracellular compartments, structurally characterize the enzyme delta 7-sterol reductase defective in the Smith-Lemli-Opitz syndrome and investigate the pathomechanism of sterol depletion-induced dysmorphogenesis.

Catalysis↗
PubMed1998

Mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome.

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 Delta7-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 fluorescense 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.

Amino Acid Sequence↗
PubMed1998

Association of calcium channel alpha1S and beta1a subunits is required for the targeting of beta1a but not of alpha1S into skeletal muscle triads.

The skeletal muscle L-type Ca2+ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca2+ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of alpha1S-beta1a interactions in the incorporation of the functional channel complex into the triad, alpha1S and beta1a [or a beta1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. betaGFP expressed in dysgenic myotubes that lack the skeletal muscle alpha1S subunit was diffusely distributed in the cytoplasm. On coexpression with the alpha1S subunit betaGFP distribution became clustered and colocalized with alpha1S immunofluorescence. Based on the colocalization of betaGFP and alpha1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of alpha1S with and without beta1a restored Ca2+ currents and depolarization-induced Ca2+ release. The translocation of betaGFP from the cytoplasm into the junctions failed when betaGFP was coexpressed with alpha1S mutants in which the beta interaction domain had been altered (alpha1S-Y366S) or deleted (alpha1S-Delta351-380). Although alpha1S-Y366S did not associate with betaGFP it was incorporated into the junctions, and it restored Ca2+ currents and depolarization-induced Ca2+ release. Thus, beta1a requires the association with the beta interaction domain in the I-II cytoplasmic loop of alpha1S for its own incorporation into triad junctions, but stable alpha1S-beta1a association is not necessary for the targeting of alpha1S into the triads or for its normal function in Ca2+ conductance and excitation-contraction coupling.

Animals↗
PubMed1998

Differential effects of Ca2+ channel beta1a and beta2a subunits on complex formation with alpha1S and on current expression in tsA201 cells.

To study the interactions of the alpha1S subunit of the skeletal muscle L-type Ca2+ channel with the skeletal beta1a and the cardiac beta2a, these subunits were expressed alone or in combination in tsA201 cells. Immunofluorescence- and green fluorescent protein-labeling showed that, when expressed alone, beta1a was diffusely distributed throughout the cytoplasm, beta2a was localized in the plasma membrane, and alpha1S was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER). Upon coexpression with alpha1S, beta1a became colocalized with alpha1S in the ER. Upon coexpression with beta2a, alpha1S redistributed to the plasma membrane, where it aggregated in large clusters. Coexpression of alpha1S with beta1a but not with beta2a increased the frequency at which cells expressed L-type currents. A point mutation (alpha1S-Y366S) or deletion (alpha1S-Delta351-380) in the beta interaction domain of alpha1S blocked both translocation of beta1a to the ER and beta2a-induced translocation of the alpha1S mutants to the plasma membrane. However, the point mutation did not interfere with beta1a-induced current stimulation. Thus, beta1a and beta2a are differentially distributed in tsA201 cells and upon coexpression with alpha1S, form alpha1S. beta complexes in different cellular compartments. Complex formation but not current stimulation requires the intact beta interaction domain in the I-II cytoplasmic loop of alpha1S.

Animals↗