Abstract
Voltage-dependent Ca(2+) channels are structurally and functionally diverse. As Ca(2+) currents recorded from embryonic chick dorsal root ganglion (DRG) neurons differ significantly from their mammalian counterparts, information on the primary sequence of the chick channels will help define the structural underpinnings of Ca(2+) channel function. Here, we report the cloning and functional expression of full-length Ca(2+) channel alpha(1B) subunit cDNAs derived from chick DRGs. Two variable regions (A and B) have been identified in the cytoplasmic linker between repeats I and II; a third (C) in the carboxyl terminus extends the open reading frame by 525 nucleotides. The A and C inserts are absent, and the B insert is present in all other class B clones reported to date. The unique shorter channels appear to predominate in DRG neurons. Results represent a requisite first step in defining the structural elements that underlie variations in function and modulation of Ca(2+) channels.
Highlights
Providing a physiological rationale for such apparent redundancy is an important challenge in the Ca2ϩ channel field
Ca2ϩ currents in embryonic chick dorsal root ganglion (DRG)1 neurons differ in significant ways from those in other species
Preparation and Screening of Chick Neuronal cDNA Library—Dorsal root ganglion neurons were dissected from 11- or 12 day-old chick (Gallus gallus) embryos (Spafas, Norwalk, CT) as described [8], and total RNA was prepared with a phenol/guanidinium thiocyanate method (Tel-Test Inc., Friendswood, TX)
Summary
Preparation and Screening of Chick Neuronal cDNA Library—Dorsal root ganglion neurons were dissected from 11- or 12 day-old chick (Gallus gallus) embryos (Spafas, Norwalk, CT) as described [8], and total RNA was prepared with a phenol/guanidinium thiocyanate method (Tel-Test Inc., Friendswood, TX). To assemble clone cdB3 (containing the longer carboxyl terminus but lacking inserts A and B in the I–II linker), cdB1-pcDNA3.1(ϩ) was cut with XbaI (70 bp downstream of KpnI on the vector) and HindIII (21 bp upstream to the end of 3Ј-untranslated region), followed by treatment of the Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA) and self-ligation. This eliminated the KpnI site at the 3Ј end of the insert. Toxin was stored and diluted as described above but delivered through a pressurized, linear array of polymer-coated quartz tubes (140 m inner diameter) positioned in the vicinity of the recorded cell
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