Abstract
The dorsal root ganglia (DRG) contain a variety of mechanoreceptors, but no molecular markers uniquely identify specific mechanoreceptor subtypes. We have used DNA microarrays and subtracted cDNA libraries to isolate genes that are specifically expressed by one type of mouse mechanoreceptor. The T-type calcium channel Cav3.2 was exclusively expressed in the DRG by D-hair receptors, a very sensitive mechanoreceptor. Pharmacological blockade of T-type calcium channels indicated that this channel may be essential for normal D-hair receptor excitability including mechanosensitivity. This is the first evidence that a calcium channel is required for normal function of a vertebrate mechanoreceptor.
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Acknowledgements
We thank A. Kanehl and H. Thränhardt for technical assistance. We thank the following individuals for help, advice and discussion: N. Hübner, F. Rathjen, T. Willnow, H. Schulz and C. Stucky. C.M.S. was supported by a Marie Curie fellowship from the European Union. This work was supported by the Deutsche Forschungsgemeinschaft and German National Genome Network grants to G.R.L. We are grateful to Roche for kindly donating mibefradil for these studies.
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The authors Jung-Bum Shin and Gary R. Lewin have filed a patent application based on some of the results described in this paper.
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Supplementary Fig. 1.
Overview of microarray and library based screen with genes identified.The strategy for screening for differentially expressed transcripts from wild type and NT-4 knockout DRG is outlined.Note that we carried out real time PCR experiments with all of the 22 genes showing a specific in situ hybridization signal in the DRG.However,for four of these genes the real time PCR primers chosen did not amplify a product (see also Excel data sheet below). (JPG 148 kb)
Supplementary Table 1.
This is a list of the 189 genes that we selected as consistently down-regulated.The criterion for selection was that reduced intensity signal of at least 1.4 fold was seen in at least two of the three experiments (3x WT vs old NT-4 –/– and 3x young vs old NT-4 –/– )a total of 9 comparisons.The absolute intensities and fold change for each of the three experiments is shown.Genes colored blue represent the 28 genes that were also present in the subtractive cDNA library.For each of these genes a description of the results of further experiments is also shown (in situ hybridization pattern and real time PCR results).In addition 10 genes colored red are annotated as these were also cloned and their DNA probes used to carry out in situ hybridization experiments on wild type and NT-4 –/– DRGs.None of these showed a D-hair receptor specific expression pattern. The excel datasheet is organized as follows.The results of each of the three microarray experiments are listed separately as experiment 1 to 3.Within each experiment the regulated genes are separated according to which of the three chips they were found on (Chips A,B,C).The absolute intensity values for each gene in wild type (NT4 wt intensity),NT-4 young knockouts (NT4-/-5w intensity)and older knockouts (NT4-/-12w intensity)is listed under the columns labeled absolute analysis.To the right of this a comparitive analysis is shown for each gene and the column header is labeled Comparison Analysis.Two camparisons are shown,the first is between wild type and older NT-4 knockouts (NT4-/-12w vs wt)and the second is between young NT-4 and old NT-4 knockouts (NT4-/-12w vs 5w).For each comparison the mean fold change and intensity difference is shown.To the right of the spreadsheet one column before the gene description the mean intensities from all three experiments with their standard deviations are shown.Finally the last three columns show the results of further experiments carried out with a subset of the genes not shown in Table 1 of the main text.Thus a summary of the results obtained from real time PCR experiments when successful and additional a brief description of the results from in situ hybridization experiments where appropriate are shown. (XLS 121 kb)
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Shin, JB., Martinez-Salgado, C., Heppenstall, P. et al. A T-type calcium channel required for normal function of a mammalian mechanoreceptor. Nat Neurosci 6, 724–730 (2003). https://doi.org/10.1038/nn1076
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DOI: https://doi.org/10.1038/nn1076
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