Research reportmRNA distribution analysis of human TRPC family in CNS and peripheral tissues
Introduction
Transient receptor potential channels are members of a very large family of cation channels composed of tetrameric assemblies of 6 transmembrane spanning subunits. The 21 member TRP family is further subdivided into three branches, the TRPC (or short TRP) sub-family, the TRPM (or long TRP) sub-family and the TRPV (or vanilloid receptor) sub-family. The TRPC sub-family, in common with all family members, are Ca2+ permeable and when activated permit Ca2+ entry at resting membrane potentials. A growing body of evidence suggests these proteins contribute to Ca2+ influx evoked by activation of the phospholipase C pathway and/or by Ca2+ store depletion [25], [21], [1], [2], [17]. However, the precise molecular composition of the channels responsible for the corresponding currents seen in native cells remains undetermined.
The first step in the molecular identification of the store-refilling pathway came from the study of phototransduction in Drosophila melanogaster. Three photoreceptor cell-specific gene products have been isolated, which have been implicated to function as Ca2+ entry channels. These are, in order of their discovery, the transient receptor potential (trp) channel [18], [34], the transient receptor potential-like channel (trpl) [24] and the transient receptor potential gamma channel (trpγ) [36]. Heterologous expression of trp in eukaryotic cells led to the formation of ion channels that could be activated by the depletion of intracellular stores [31], [22]. Moreover heterologous expression of trpl and trpγ produced currents that were seemingly not store-operated but instead exhibited constitutive activity [7], [10]. To date, seven mammalian trpc gene homologues of drosophila TRP channels have been identified, termed TRPC1-TRPC7.
TRPC1, the first member of the mammalian TRPC family to be characterised, appears to produce a current in response to intracellular Ca2+ store depletion [40], [19]. TRPC2, also, has been shown to be activated subsequent to depletion of intracellular Ca2+ stores [32], although the human orthologue TRPC2 has an early stop codon and appears to be a pseudogene [33]. Furthermore, TRPC3, TRPC4, TRPC5 and TRPC7 have been implicated in both receptor- and store-operated channel function, depending on both the expression system used and the species studied (i.e. human or mouse) [38], [41], [23], [27], [20], [26], [3]. Lastly, TRPC6, and TRPC3, expressed in recombinant systems, are activated by diacylglycerol independent of the ability of this lipid to activate protein kinase C [9].
TRPCs seem to be ubiquitously expressed in mammalian tissues. However, previous reports have described only limited data on the mRNA distribution of these channels in human tissues, obtained with techniques such as Northern blotting and RT-PCR.
In this report we describe the first detailed and comparative mRNA distribution study of the TRPC family in a wide range of human tissues and cells. We have utilized the technique of TaqMan RT-PCR to more accurately compare the distribution profiles of this family of channels in a similar way to that described recently for the two pore potassium channel family [16].
Section snippets
RNA and cDNA synthesis
Poly A+ RNA from multiple tissues of four different individuals (two men, two females except prostate), were provided either by Dr. R. David of the Netherland’s Brain Bank, Amsterdam or purchased as prepared RNA from Biochain (San Leandro, CA) or Clontech (Palo Alto, CA). All samples were donor-anonymous and were obtained under conditions of informed consent. Total RNA from cell lines was prepared using the Trizol reagent (Invitrogen, Groeningen, The Netherlands) following the manufacturer’s
Results
Gene-specific TaqMan primers and probe sets were designed and preliminary experiments showed them to amplify specifically and efficiently for each channel.
TaqMan RT-PCR analysis of the human TRPC1, TRPC3, TRPC4, TRPC5 TRPC6 and TRPC7 channels was carried out in CNS and peripheral tissues, as well as a number of cell lines. mRNA samples viability was previously assessed using the housekeeping genes cyclophilin and β-actin. Minimal variation in the expression of these genes was observed between
Discussion
The TRPC family is a structurally defined group of Ca2+-permeable non-selective cation channels that are expressed throughout the CNS and in many peripheral tissues. To date seven human TRPCs have been cloned. Although a number of previous distribution studies have documented the expression of these channels in certain human tissues, the present study is the first to systematically quantify their expression in a broad range of human tissues, including a host of discrete brain areas.
Acknowledgements
We thank Dr Paul Murdock for providing Taqman tissue plates and Jean-Philippe Walhin for technical assistance; Dr Martine Garnier for valuable comments on the manuscript. We also thank Dr R. Ravid of The Netherland’s Brain Bank for the arrangement/donation of brain tissue. Antonio Riccio and Cesar Mattei are in receipt of EU Framework V Postdoctoral Fellowships.
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