β1B subunit of voltage‐dependent Ca²⁺ channels is predominant isoform expressed in human neuroblastoma cell line IMR32

Abstract

Human neuroblastoma cells (IMR32) respond to treatment with either dibutyryl-cAMP or nerve factor by acquiring a neuronal phenotype which is accompanied by a marked increase in the density of neuronal (N-type) VDCC currents. Using IMR32 cells as a model for neuronal differentiation, we were interested in examining possible changes in the level of expression of the K K1B subunit of N-type calcium channels as well as beta subunit isoforms. Upon differentiation with dibutyryl-cAMP and 5-bromo-2-deoxyuridine for 16 days, we observed a dramatic increase in K K1B protein which initiated between day 8 and 10. Day 10 evidenced maximal expression of K K1B protein, which was followed by an interval of relatively constant expression of K K1B (day 12 to day 16). Monitoring beta subunit expression using a pan specific anti-beta antibody (Ab CW20), we observed an increase in expression of a single 82 kDa beta subunit. The predominant 82 kDa beta subunit expressed throughout the course of differentiation was identified as the L L1b isoform using a panel of beta subunit specific antibodies. Of significance, neither the L L2 nor L L3 isoforms were detected in full differentiated IMR32 cells. Contrary to a previous report on the absence of neurotypic expression of VDCC beta subunits in a second model for in vitro differentiation, NGF-treated rat pheochromocytoma cells (PC12 cells) [1], we report the regulated expression of the L L1b protein in differentiated IMR32 cells suggesting a cell specific function for this beta subunit which parallels the acquisition of the neuronal phenotype. The restrictive expression of the L L1b in IMR32 cells may reflect a cell-type specific function that extends beyond its role as an auxiliary subunit of VDCC complexes.z 1997 Federation of European Biochemical Societies.

Differential Expression and Association of Calcium Channel α1B and β Subunits during Rat Brain Ontogeny

“…The coupled peptide antigens were used in the production of polyclonal sera in rabbits under continued contractual agreement with Covance, Inc. The rabbits were bled twice per month (15)(16)(17)(18)(19)(20) ml/bleed) and tested for production of specific antibodies after 4 weeks as described previously (34).…”

“…Polyclonal antibodies were affinity purified on antigen affinity columns as described (44). The ␤ subunit isoform-specific antibodies have been characterized elsewhere (23,34). Immunoprecipitation of N-type VDCC-The N-type VDCC was solubilized from P2 brain and P14 and adult rat forebrain as described previously (21) with the following modifications: the N-type VDCC from P2 brain and P14 forebrain were solubilized directly from membranes using 0.75% CHAPS.…”

“…Several families of ligand-gated ion channels evidence developmental changes in their subunit composition (27)(28)(29). Whereas studies have identified alterations in the expression of N-type and P/Q-type VDCC during synapse formation in cultured neurons (30 -32), few reports have investigated the developmental or differentiation-dependent expression of calcium channel subunits and their assembly (33,34). Interestingly, there have been reports of N-type VDCC in developing cerebellar granule cells (35) and differentiated human neuroblastoma SH-SY5Y cells (36) which are inhibited by -conotoxin GVIA (CTX) yet display two components of inactivation.…”

“…The first antibody, Ab CW14, was raised to an epitope present in all ␣ 1B subunits cloned to date (34). The second antibody, Ab CW8, was raised to an epitope present only in the rat ␣ 1B sequence (37).…”

A Syntaxin 1, Gαo, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

“…Mouse monoclonal antibodies used in this study were: G␣ O, G␣ O AB-1 (Lab Vision, Freemont, CA); syntaxin (Sigma, St. Louis, MO); G␤, MUNC18 (BD Transduction Lab, San Diego, CA), and SV2, Na/K pump (Developmental Studies Hybridoma Bank, Iowa City, IA). Affinity-purified polyclonal antibodies were: neurofilaments, AB1991 (Chemicon, Temecula, CA); MUNC18 (ABR); syntaxin (Sigma); SV2 (Stressgen, Vancouver, British Columbia, Canada); Ca V 2.1, CW65 (Fletcher et al, 2001), Ca V 2.2, CW21 (also termed CW14; McEnery et al, 1997). CW65 and CW21 were gifts from M. McEnery (Case Western Reserve University, Cleveland, OH).…”