Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains

Abstract

Background: The purpose of this work was to study the gene expression of transient receptor potential (TRP) channels in the mouse. The application of a standardized and quantitative technique, TaqMan RT-PCR, should give information about the pattern and relative importance of TRP channels for murine tissues and cell types. To verify data sets with an independent method, we studied the occurrence of some of the transcripts by in situ hybridization.

Results: We have characterized the mRNA expression of 22 TRP channels in the mouse with a focus on nerve and muscle tissues. This is the first study to describe the expression profiles of all channel isoforms of the four related Group 1 subfamilies (TRPC, TRPV, TRPM and TRPA) with a standardized and quantitative technique. Comparisons of transcript abundance showed a consistent dominance of TRPM7 and TRPC3 in most tissues. We further observed characteristic patterns and differences in gene expression of individual channels ranging over three orders of magnitude. The overall level of TRP channel mRNAs was highest in brain areas followed by kidney, lung, reproductive organs and muscle. In brain TRPM3 and TRPM7 dominated and 19 other isoforms were detected. In lung and kidney TRPV4, TRPV5 and TRPM7 were found in highest levels. TRPM7, TRPC3, TRPC6 and TRPM3 mRNAs were characteristically present in all tested muscle tissues. Most data obtained with the C57Bl/10 mouse strain were confirmed with Balb/c and NOD mice. However, TRPC3, C6, TRPM7, M3, TRPV2 and V4 expression showed marked differences in the three tested mouse strains. In situ hybridization revealed co-expression of transcripts on the cellular level and widely confirmed the data obtained with RT-PCR.

Conclusion: Transcripts coding for members of the TRPC, TRPV, TRPM and TRPA subfamilies of TRP cation channels are present in a broad spectrum of murine tissues. Several channel isoforms often coexist in a specific tissue or cell type. TRP channel expression does not show typical tissue specific dominance of individual members as is known from other ion channel families. Mouse strain specific variations of TRP channel expression indicate that genetic background or physiological requirements considerably influence expression levels.

Figure 1

Expression of TRP channel mRNAs in murine tissues using standard RT-PCR. Reverse transcribed RNA (8 ng) from tissues of adult (100 d) C57Bl/10SC mice was added to the reaction mixtures and PCR products amplified in 40 cycles. The products were separated on agarose gels and stained with ethidium bromide. Water served as negative control. 1) testis; 2) liver; 3) lung; 4) spleen; 5) skeletal muscle; 6) epididymis; 7) kidney; 8) ovary; 9) seminal vesicle; 10) uterus; 11) C2C12 cells; 12) H₂O; 13) aorta; 14) intestine; 15) heart; 16) hippocampus; 17) brain stem 18) forebrain; 19) cerebrum 20) cerebellum; 21) truncus encephali.

Figure 2

Quantification of TRP ion channel mRNA levels in murine tissues using Real-time RT-PCR. Expression of TRPC1-7 (A), TRPV1-6 and TRPA1 (B) as well as TRPM1-8 (C) mRNA was analyzed in murine tissues using Real-time RT-PCR. Samples were from adult (100 d) C57Bl/10SC mice. The mRNA levels of TRP ion channels are given in relation to 18S rRNA. Means ± S.E.M. are given in all cases for n = 3–10 samples.

Figure 3

Summary of tissue specific TRP channel gene expression. Expression of TRPC1-7, TRPM1-8, TRPV1-6 and TRPA1 mRNAs in murine tissues was determined by real-time RT-PCR. Levels of mRNAs are given in relation to that of 18S rRNA; all data were multiplied by 1000. Means were calculated from n = 3–10 samples and classified into 5 categories. If the threshold level of fluorescence according to the TaqMan RT-PCR technique was not reached after 40 cycles of amplification, a resulting expression level of zero (0) was assigned to the corresponding channel transcript and tissue. For better illustration categories of gene expression levels are represented by different colors ranging from zero (white) to values higher than 10 (dark blue).

Figure 4

In situ localization of three TRPC mRNAs in different mice organs using in situ hybridization. In situ hybridization of TRPC3, TRPC5 and TRPC6 mRNA was performed with tissue sections of adult (100d) C57Bl/10SC mice. The two left hand columns of sections were incubated with the antisense probe for TRPC3 and the sense probe (control), respectively. For TRPC5 and TRPC6 only results with antisense probes are shown. Tissue sections: A: hippocampus, B: Kidney, C: Lung, D: epididymis, E: skeletal muscle. In all cases a methyl green counter-staining was performed. Magnification: × 200.

Figure 5

Comparison of TRP channel mRNA levels in different mouse strains. Expression of TRPC3, C6, M3, M7, V2 and V4 channels was determined by Real-time RT-PCR in selected tissues of three different mouse strains. Tissue samples were collected from adult (100 d) C57Bl/10SC (grey columns), NOD (black columns) and Balb/c mice (dotted columns). The mRNA levels of TRP channels are given in relation to 18S rRNA for cerebrum (A), kidney (B) skeletal muscle (C) and heart (D). Means ± S.E.M. are given in all cases for n = 3–10 samples.