J Periodontal Implant Sci.  2015 Apr;45(2):69-75. 10.5051/jpis.2015.45.2.69.

Multiple transcripts of anoctamin genes expressed in the mouse submandibular salivary gland

• Han JH
• Kim HM
• Seo DG
• Lee G
• Jeung EB
• Yu FH

• KMID: 2329658
• DOI: http://doi.org/10.5051/jpis.2015.45.2.69

Abstract

PURPOSE
Salivary fluid formation is primarily driven by Ca2+-activated, apical efflux of chloride into the lumen of the salivary acinus. The anoctamin1 protein is an anion channel with properties resembling the endogenous calcium-activated chloride channels. In order to better understand the role of anoctamin proteins in salivary exocrine secretion, the expression of the ten members of the anoctamin gene family in the mouse submandibular gland was studied.
METHODS
Total RNA extracted from mouse submandibular salivary glands was reverse transcribed using primer pairs to amplify the full-length coding regions of each anoctamin gene and was subcloned into plasmid vectors for DNA sequencing. Alternative splice variants were also screened by polymerase chain reaction using primer pairs that amplified six overlapping regions of the complementary DNA of each anoctamin gene, spanning multiple exons.
RESULTS
Multiple anoctamin transcripts were found in the mouse submandibular salivary gland, including full-length transcripts of anoctamin1, anoctamin3, anoctamin4, anoctamin5, anoctamin6, anoctamin9, and anoctamin10. Exon-skipping splicing in the N-terminal exons of the anoctamins1, anoctamin5, and anoctamin6 genes resulted in multiple alternative splice variants. No expression of anoctamin2, anoctamin7, or anoctamin8 was found.
CONCLUSIONS
The predominant anoctamin transcript expressed in the mouse submandibular gland is anoctamin1ac. The chloride channel protein produced by anoctamin1ac is likely responsible for the Ca2+-activated chloride efflux, which is the rate-limiting step in salivary exocrine secretion.

Keyword

Alternative splicing; Chloride channels; Submandibular gland

MeSH Terms

Alternative Splicing
Animals
Chloride Channels
Clinical Coding
DNA, Complementary
Exons
Humans
Mice*
Plasmids
Polymerase Chain Reaction
RNA
Salivary Glands*
Sequence Analysis, DNA
Submandibular Gland
Chloride Channels
DNA, Complementary
RNA

Figure

• 

  Figure 1 Full-length anoctamin cDNA sequences reverse-transcribed from mouse submandibular gland total RNA. (A,B) Specific primers were designed to amplify the entire coding regions of all 10 members of the anoctamin gene family. RT-PCR products were size fractionated on 0.8% agarose gel. Arrows indicate full-length amplicons. M, DNA ladder; kbp, kilobase pair.

• 

  Figure 2 Splicing of anoctamin1 transcripts in the mouse submandibular gland. (A) Anoctamin1 gene organization showing 26 exons. Six PCR primer pairs were designed to amplify overlapping regions of anoctamin1, spanning multiple exons. (B) Size fractionation of RT-PCR products of anoctamin1. Arrow head indicate an additional 570 bp band amplified between exons 2 and 9. (C) The location of splice site exon 7 which is deleted in anoctamin1ac. E, exons; TM, transmembrane segment.

• 

  Figure 3 Splicing of anoctamin5 transcripts in the mouse submandibular gland. (A) Anoctamin5 gene organization showing 22 exons. Six PCR primer pairs were designed to amplify overlapping regions of anoctamin5, spanning multiple exons. (B) Size fractionation of RT-PCR products of anoctamin5. Arrowheads indicate additional bands: approximately 330 bp band between E1 and E7 (anoctamin5a); approximately 1 kb and 950 bp bands between E1 and E12 (anoctamin5a and anoctamin5b); approximately 500 bp band between E9 and E15 (anoctamin5b); approximately 500 bp and 400 bp bands between E14 and E18 (anoctamin5c); and approximately 700 bp, 530 bp, and 430 bp bands between E18 and E22 (anoctamin5d, anoctamin5e, and anoctamin5f). (C) Location of alternatively spliced exons of anoctamin5.

• 

  Figure 4 Splicing of anoctamin6 transcripts in the mouse submandibular gland. (A) Anoctamin6 gene organization showing 20 exons. Six PCR primer pairs were designed to amplify overlapping regions of anoctamin6, spanning multiple exons. (B) Size fractionation of RT-PCR products of anoctamin6. Arrowheads indicate additional bands: approximately 880 bp and 500 bp bands between E1and E7 (anoctamin6a, anoctamin6b); approximately 420 bp band between E4 and E9 that is further diagnostic of anoctamin6b; and an approximately 440 bp band between E17 and E22 (anoctamin6c). (C) The location of the alternatively spliced exons of anoctamin6.

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