Elsevier

Molecular Brain Research

Volume 43, Issues 1–2, 31 December 1996, Pages 259-266
Molecular Brain Research

Research report
Cloning and expression localization of cDNA for rat homolog of TRP protein, a possible store-operated calcium (Ca2+) channel

https://doi.org/10.1016/S0169-328X(96)00208-2Get rights and content

Abstract

A 3.2 kbp cDNA clone encoding a possible candidate for the store-operated Ca2+ channel was isolated from a rat brain cDNA library. The deduced amino acid sequence was 51.8% identical to TRP encoded by a Drosophila trp (transient receptor potential) gene and contained ankyrin motifs, a coiled-coil structure and six transmembrane segments similar to the previously identified TRP family and named as TRP-R (rat TRP). By in situ hybridization histochemistry of rat body on embryonic day 15, no significant expression signal for TRP-R was detected. On embryonic day 20 and postnatal day 1, the expression signals were most evident in the septum, cerebral cortical plate and hippocampal neuronal layers. On postnatal day 7 and thereafter the expression in the cerebral cortex and the septum decreased progressively, and weak expression remained only in the septum and CA1 and CA2 neuronal layers of the hippocampus in the brain on postnatal day 21 and 49. This limited spatiotemporal expression of this novel molecule, TRP-R, suggests that it is involved in some specific functions related to the neuronal differentiation.

Introduction

In terms of intracellular Ca2+ signaling, cells are categorized into excitable and non-excitable ones, although there has recently been evidence that Ca2+ signaling mechanisms in excitable and non-excitable cells are more similar than previously thought [16]. Both cells utilize Ca2+ released from intracellular storing organelles such as the endoplasmic reticulum or the `calciosomes' for signaling. The predominant mechanism for release is triggered by a diffusible messenger, inositol 1,4,5-triphosphate(IP3) in non-excitable cells, while the CICR(Ca2+-induced Ca2+ release) mechanism operates for Ca2+ release in excitable cells [5]. Non-excitable cells exhibit a biphasic process in the Ca2+ signaling: In the first phase, binding of plasma membrane receptors with neurotransmitters or hormones activates enzymes generating IP3 from plasma membrane phospholipids. The liberated IP3 binds with and stimulates its specific receptors on the intracellular Ca2+-storing organelle to release Ca2+ into the cytoplasm. In the second phase the empty Ca2+-storing organelle produces a supposed retrograde signal that activates Ca2+ influx across the plasma membrane from the extracellular space. This process is referred to as store-operated capacitative Ca2+ entry. An electrical current associated with this influx has been characterized and designated Ca2+ release-activated Ca2+ current (ICRAC) and the channel responsible for this current is known as CRAC channels or store-operated Ca2+ channel (SOC) 9, 10, 12.

There has been suggestion that one of the most likely candidates for CRAC channel or SOC is a mammalian homolog for the Drosophila protein termed TRP. Drosophila mutant lacking trp shows a transient receptor potential (trp) in the photoreceptors in response to light, which is suggested to be due to the absence of store-operated Ca2+ entry [14]. Molecular characterization of the trp gene has revealed a predicted protein of 1275 amino acids with six putative transmembrane domains, an ankyrin-like repeat domain and a calmodulin-binding site 14, 15, 19. Subsequently, screen for calmodulin-binding protein in the Drosophila head has identified a protein with a significant sequence similarity to the TRP protein in the photoreceptors and termed TRPL (TRP-like) [15].

On the assumption that there is a significant homology in amino acid sequences between TRP/TRPL and mammalian counterparts, the present study was undertaken to isolate and sequence cDNAs encoding mammalian TRP proteins. During this attempt Wes et al. have cloned a cDNA for a human homolog of trp, termed TRPC1, richly expressed in fetal brain, as well as adult brain, heart, testis and ovary [18]. However, no detailed information was available on its spatiotemporal localization in developing and mature brains, which would represent in general the first important information for understanding the functional role of given molecules.

In this paper we reported the isolation and sequencing of cDNA encoding a trp homolog of rat, which is in general a system of best choice for detailed localization of given molecules at levels of mRNA as well as protein, and the gene expression for this novel molecule was revealed to be confined largely to the septum and cerebral cortex at perinatal stages.

Section snippets

cDNA library, PCR and hybridization screening

Total RNA was extracted from rat brain on the postnatal day 21 (P21) by guanidine thiocyanate/phenol/chloroform extraction [4]. Poly(A) RNA was isolated by chromatography on an oligo(dT) cellulose column. Primers used for PCR (polymerase chain reaction) were composed of two degenerate oligonucleotides which were synthesized according to the amino acid sequences of conserved regions between Drosophila TRP and TRPL and the sequences for the primers were designed according to the mammalian codon

Nucleotide sequence and primary structure

Fig. 1 shows the restriction enzyme map, the nucleotide sequence of a cloned cDNA and its deduced amino acid sequence. The cDNA insert contained a single open reading frame of 3290 bp encoding 941 amino acids, from which the molecular mass was calculated to be 107 755.

The identity in the amino acid sequence between this novel molecule and the previous TRP family proteins was significantly high: 42.1% identity with Drosophila TRP over 470 amino acids; 51.8% identity with TRPL over 365 amino

Discussion

The present study identified a cDNA encoding a novel molecule sharing high identity in the putative transmembrane domain (64%) and both remainder C- and N-terminal domains (60% and 72%, respectively) to Drosophila TRP and TRPL 14, 15, 19, and this molecule is thus termed TRP-R (rat homolog of TRP). This high homology, together with the absence of charged residues, suggests that TRP-R is a possible candidate for the mammalian store-operated Ca2+ channel (SOC), although functional expression

Acknowledgements

This study was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, Nos. 07044216, 07457001, 07278203 to H.K. and No. 07780670 to K.G., and also by a grant from the Tanaka Orthopedic Hospital Foundation, Hamamatsu, Japan.

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