Research reportDistribution of the organellar Ca2+ transport ATPase SERCA2 isoforms in the cat brain
Introduction
Excitatory synaptic transmission in the brain is triggered by depolarization of the presynaptic membrane, followed by an increase of the cytosolic Ca2+ level in the presynaptic terminal and release of neurotransmitter, usually glutamate. An excitatory transmitter in its turn may induce depolarization of the postsynaptic membrane, the production of the messenger inositol 1,4,5-trisphosphate (InsP3), and an increase of the cytoplasmic Ca2+ concentration in the postsynaptic region. While the depolarization might be sufficient to carry the message of excitation along the membrane of the neurons, Ca2+ is crucial for short-term and long-term changes of synaptic transmission, and probably is involved in learning and memory [8]. The major systems involved in the regulation of neuronal Ca2+ are Ca2+ channels of the plasma membrane, Ca2+-release channels of the internal Ca2+ stores (InsP3 receptors and ryanodine receptors), Ca2+ buffering proteins, the Na+/Ca2+ exchanger, and ATP-dependent Ca2+ transport ATPases (Ca2+ pumps).
The Ca2+ pumps of the internal Ca2+ stores belong to the SERCA (Sarco(Endo)plasmic Reticulum Calcium transporting ATPase) gene family, which consists of three members: SERCA1, SERCA2, and SERCA3. Among these different isoforms, SERCA2 seems to be the major, if not the only isoform present in most brain regions. Indeed, SERCA1 has never been detected in the brain and the SERCA3 expression was found to be restricted to the Purkinje neurons of the cerebellum 1, 24.
Alternative splicing of the primary transcript of the SERCA2 gene gives rise to two protein isoforms: SERCA2a and SERCA2b. These isoforms differ in their carboxyl terminal end as the last four amino acids of SERCA2a are replaced by a stretch of 49 amino acids in SERCA2b. SERCA2a is expressed in cardiac, slow skeletal and to some extent also in smooth muscle, whereas SERCA2b is ubiquitously expressed 4, 7, 10. SERCA2b shows a twofold higher affinity for Ca2+ but a twofold lower catalytic turnover rate than SERCA2a [23].
The expression of the SERCA2 gene in the rat brain has been assessed at the mRNA level using probes which do not discriminate between the SERCA2a and SERCA2b messengers [11]. With the exception of a study on the Purkinje neurons of the pig [15]and avian cerebellum [3], nothing is known on the distribution of SERCA2 Ca2+ pump isoforms at the protein level in the brain. In this paper we have made a detailed study of the localization of SERCA2a/SERCA2b in the cat brain using immunocytochemistry. To confirm the specificity of our results, in situ hybridization has been carried out in parallel. We have chosen the cat with the view on future experiments on the effects of visual training of cats on the expression of proteins involved in Ca2+ metabolism. In addition we have compared the distribution of SERCA2 in the cat brain with that in the rat.
Section snippets
Animal and tissue processing
Adult cats or rats were deeply anesthetized with Ketalar. Adult cats were subsequently killed by an overdose of Nembutal. The brain was perfused in situ, via the carotid arteries, with ice cold 50 mM Tris, 0.9% NaCl, pH 7.4 (TBS) followed by 4% paraformaldehyde in 100 mM sodium phosphate buffer, pH 7.4 (PB).
For in situ hybridization (ISH), different parts of the cat brain were then excised, immersed in methyl-butane and frozen in liquid nitrogen. Sections of 15 μm thickness were cut on a
Western blot analysis
The presence of SERCA2a or SERCA2b was first tested by immunoblotting of microsomal protein obtained from the cat cerebrum, cerebellum, and brainstem. No reaction could be visualized with the SERCA2a antibody when applying a concentration of 100 μg of microsomal protein per lane (Fig. 1A). In contrast, SERCA2b gave a strong signal and stained a single band at the expected size (100 kDa) in all three microsomal brain fractions (Fig. 1B).
Immunocytochemistry: general considerations
Tracings of the sections were made from the projection of
Discussion
Although it has been shown by ISH and Northern blotting that the SERCA2b isoform is the predominant isozyme of the organellar-type Ca2+ transport ATPases expressed in the mammalian brain, no detailed study on the localization of the SERCA2b/SERCA2a protein in the brain has been published. This paper describes the distribution of the SERCA2 Ca2+ transport ATPases in the cat brain as revealed by SERCA2a- and SERCA2b-specific antibodies.
Acknowledgements
We thank the members of the laboratory of Prof. F. Vandesande (Neuroendocrinology and Immunological Biotechnology, KULeuven, Belgium) and of Prof. G. Orban (Neuro-and Psychophysiology, KULeuven, Belgium), I. Willems, Y. Parijs and R. Verbist (Physiology, KULeuven) for their practical help. We also thank Prof. M. Celio (Institute of Histology, Faculty of Sciences, Pérolles, Fribourg, Switzerland) and Dr. F. Viana (Physiology, KULeuven) for the fruitful discussions in interpreting the results.
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