Expression of Ca2+ channel subunits during cardiac ontogeny in mice and rats: Identification of fetal alpha1C and beta subunit isoforms

Functional cardiac L-type calcium channels are composed of the pore- forming {a1C) subunit and the regulatory {beta}2 and {alpha}2/{delta} subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of {alpha}(1C) and {beta}2 subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of {alpha}(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa {alpha}(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa {alpha}(1C) decreased to undetectable levels. The expression profile of the 85-kDa {beta}2 subunit was completely different: {beta}2 was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional {beta}2-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of {beta}2 subunit variants. Furthermore, two fetal proteins with {beta04 immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, {beta}{beta}4 gene expression was confirmed by RT-PCR. Cardiac and brain {beta}4 mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca2+ channel subunits and suggest a role of fetal {alpha}(1C) and {beta} isoforms in the assembly of Ca2+ channels in immature cardiomyocytes

Identification of osmo-dependent and osmo-independent betaine-choline-carnitine transporters in Acinetobacter baumannii: role in osmostress protection and metabolic adaptation

Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed

Differential expression and association of calcium channel alpha1B and beta subunits during rat brain ontogeny

Calcium functions as an essential second messenger during neuronal development and synapse acquisition. Voltage-dependent calcium channels (VDCC), which are critical to these processes, are heteromultimeric complexes composed of alpha1, alpha2/delta, and beta subunits. beta subunits function to direct the VDCC complex to the plasma membrane as well as regulate its channel properties. The importance of beta to neuronal functioning was recently underscored by the identification of a truncated beta4 isoform in the epileptic mouse lethargic (lh) (Burgess, D. L., Jones, J. M., Meisler, M. H., and Noebels, J. L. (1997) Cell 88, 385-392). The goal of our study was to investigate the role of individual beta isoforms (beta1b, beta2, beta3, and beta4) in the assembly of N-type VDCC during rat brain development. By using quantitative Western blot analysis with anti-alpha1B-directed antibodies and [125I-Tyr22]omega-conotoxin GVIA (125I-CTX) radioligand binding assays, we observed that only a small fraction of the total alpha1B protein present in embryonic and early postnatal brain expressed high affinity 125I-CTX-binding sites. These results suggested that subsequent maturation of alpha1B or its assembly with auxiliary subunits was required to exhibit high affinity 125I-CTX binding. The temporal pattern of expression of beta subunits and their assembly with alpha1B indicated a developmental pattern of expression of beta isoforms: beta1b increased 3-fold from P0 to adult, beta4 increased 10-fold, and both beta2 and beta3 expression remained unchanged. As the beta component of N-type VDCC changed during postnatal development, we were able to identify both immature and mature forms of N-type VDCC. At P2, the relative contribution of beta is beta1b > beta3 >> beta2, whereas at P14 and adult the distribution is beta3 > beta1b = beta4. Although we observed no beta4 associated with the alpha1B at P2, beta4 accounted for 14 and 25% of total alpha1B/beta subunit complexes in P14 and adult, respectively. Thus, of the beta isoforms analyzed, only the beta4 was assembled with the rat alpha1B to form N-type VDCC with a time course that paralleled its level of expression during rat brain development. These results suggest a role for the beta4 isoform in the assembly and maturation of the N-type VDCC