Molecular cloning of multiple subtypes of a novel rat brain isoform of the α1 subunit of the voltage-dependent calcium channel

Hui, Anna; Ellinor, Patrick T.; Križanová, Oľga; Wang, Jing-Ding; Diebold, Ronald J.; Schwartz, Arnold

doi:10.1016/0896-6273(91)90072-8

Cited by 186 publications

(104 citation statements)

References 43 publications

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“…The hybridization probe was prepared from the pGEM plasmid (p6OZ) containing a 443 bp insert (residue number 2803-3246 as in [16]) from DHPR-I3 cDNA, which encodes the cytoplasmic loop bctween the repeating unit 11 S6 and III Sl. Antisense and sense riboprobes were prepared from the p6OZ plasmid, which had been linearized with appropriate restriction enzymes, by transcribing with appropriate RNA polymerases using a Riboprobe System (Pro~ucga Corp., Madison, WI) in the presence of a-'*S-UTP (IOOO-1500 Cii mmol, New England Nuclear).…”

Section: Crn4 Probe Preparathn Artd In Siru Hybridkationmentioning

confidence: 99%

“…Analysis of these brain cDNAs further revealed that the brain al subunit isoforms with sequence similarity to the skeletal muscle Ca2+ channel, which are produced by a family of related genes [13], encode the DHP-sensitive Ca" channels [14]. We and others have isolated a novel brain isoform of the DHPsensitive Ca"' channel or1 subunit cDNA (DHPR-B in [I 51; RBal in [16]) whose primary structure is significantly different from its skeletal and cardiac muscle counterparts. Here, we describe the distribution in rat brain of the mRNA encoding this form of the DHPsensitive Ca2' channel al subunit by in situ hybridization, and that of the c&l subunit protein by immunohistochemistry.…”

Section: Introductionmentioning

confidence: 99%

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Expression of dihydropyridine‐sensitive brain calcium channels in the rat central nervous system

et al. 1992

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WC have localized dihydropyridine (DHP)-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel al subunit (DHPR-B) is prominently tocalized in neuronal cells in the olfactory bulb, dentate gyrus, hippooampus, arcuate nucleus, paraventricular nucleus, vcntromcdial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pincal glands. DHP-sensitive calcium channel 01 subunit distribution has also been examined immunohistochcmically with polyclonal antibodies raised agGnst synthetic pptides specific for the DHPR-B al subunit protein. The results from immunohistochemistry were in good aSmemcnt with those from in situ hybridization. Thus, qional distribution and localization of DHPR-B mRNA and rl subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium cbanncl may play an important role in excitation-secretion coupling functions in the neuroendocrine system.

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Section: Crn4 Probe Preparathn Artd In Siru Hybridkationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression of dihydropyridine‐sensitive brain calcium channels in the rat central nervous system

et al. 1992

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“…The resulting PCR cDNA fragments were then cloned into the pGEM EX-1 vector (Promega, Madison, W I), and the cDNA clones were confirmed by sequencing. The ␣ 1C cDNA was a 333 bp fragment (nt 3306 -3638) (Snutch et al, 1991), and the ␣ 1D cDNA was a 284 bp fragment (nt 2902-3185) (Hui et al,1991) spanning a region at the II-III linker.…”

Section: Methodsmentioning

confidence: 99%

Calcium Channel Density and Hippocampal Cell Death with Age in Long-Term Culture

et al. 1997

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The expression of voltage-gated calcium (Ca 2ϩ ) channel activity in brain cells is known to be important for several aspects of neuronal development. In addition, excessive Ca 2ϩ influx has been linked clearly to neurotoxicity both in vivo and in vitro; however, the temporal relationship between the development of Ca 2ϩ channel activity and neuronal survival is not understood. Over a period spanning 28 d in vitro, progressive increases in high voltage-activated whole-cell Ca 2ϩ current and L-type Ca 2ϩ channel activity were observed in cultured hippocampal neurons. On the basis of single-channel analyses, these increases seem to arise in part from a greater density of functionally available L-type Ca 2ϩ channels. An increase in mRNA for the ␣ 1 subunit of L-type Ca 2ϩ channels occurred over a similar time course, which suggests that a change in gene expression may underlie the increased channel density. Parallel studies showed that hippocampal neuronal survival over 28 d was inversely related to increasing Ca 2ϩ current density. Chronic treatment of hippocampal neurons with the L-type Ca 2ϩ channel antagonist nimodipine significantly enhanced survival. Together, these results suggest that age-dependent increases in the density of Ca 2ϩ channels might contribute significantly to declining viability of hippocampal neurons. The results also are analogous to patterns seen in neurons of aged animals and therefore raise the possibility that long-term primary neuronal culture could serve as a model for some aspects of aging changes in hippocampal Ca 2ϩ channel function.

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“…These features are also characteristic of the other three S2 or S3 segments due to extensive sequence homology. In addition to expression of multiple genes, diversity of calciumchannel transcripts is generated by alternative splicing, confirmed for the IVS3 region (Hui et al, 1991;Snutch et al, 1991); splice variants show differences in hydrophobic residues, whereas charged residues are conserved.…”

Section: Sequence Considerationsmentioning

confidence: 99%

Design of a functional calcium channel protein: Inferences about an ion channel‐forming motif derived from the primary structure of voltage‐gated calcium channels

et al. 1993

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To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (SI-S6) predicted to form membrane-spanning, a-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cationselective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCI, is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and SrZf are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by pM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.Keywords: calcium channel; dihydropyridines; lipid bilayer; protein design; four-helix bundle; single-channel recording Voltage-gated channels, comprising a large superfamily of integral membrane proteins fundamentally involved in electrical excitability (Hille, 1992), facilitate the flux of ions across the cell membrane in response to changes in transmembrane electric field. Because voltage-gated channel proteins have not yet yielded to high-resolution structural analysis, the unequivocal localization of even fundamental motifs, such as the actual ionic pathway, is lacking. However, understanding the unique functional attributes of channel proteins requires that structural elements underlying specific properties are identified. Extensive efforts are currently directed toward elucidating molecular determinants underlying the ionic pore.

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Molecular cloning of multiple subtypes of a novel rat brain isoform of the α1 subunit of the voltage-dependent calcium channel

Cited by 186 publications

References 43 publications

Expression of dihydropyridine‐sensitive brain calcium channels in the rat central nervous system

Expression of dihydropyridine‐sensitive brain calcium channels in the rat central nervous system

Calcium Channel Density and Hippocampal Cell Death with Age in Long-Term Culture

Design of a functional calcium channel protein: Inferences about an ion channel‐forming motif derived from the primary structure of voltage‐gated calcium channels

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