Human islets of Langerhans express the δC isoform of calcium/calmodulin-dependent protein kinase II

✍ Scribed by Lorraine Southam; Rebecca Ashfield; Roger Cox; Mark Lathrop; Stephen J.H. Ashcroft

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 98 KB
• Volume: 15
• Category: Article
• ISSN: 1520-7552
• DOI: 10.1002/(sici)1520-7560(199907/08)15:4<243::aid-dmrr40>3.0.co;2-3

✦ Synopsis

Background There is considerable evidence that calcium/calmodulindependent protein kinase II (CaM kinase II) plays a key role in insulin secretion and the enzyme provides a candidate gene for Type 2 diabetes. Since several isoforms of the enzyme exist, it is essential to de®ne which are expressed by the b-cell.

Methods A human islet cDNA library in lZAPII was screened with a probe for the 5k-end of human c CaM kinase II. Since this region is very homologous between the different isoforms, it is expected that isoforms other than c would be detected. From each of the six positive clones obtained, DNA was prepared and subjected to PCR using primers spanning the variable region in which the main variability of CaM kinase II isoforms resides. PCR products were puri®ed and sequenced in both directions. The b-cell line MIN6 was screened for CaM kinase II d by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blotting.

Results

The sequences of ®ve of the human islet PCR products indicated that the clones corresponded to the c B isoform whose expression in human islets we have previously documented. The other PCR product, however, gave a sequence containing the variable domains II and VII characteristic of CaM kinase II d. This sequence and the absence of other domains in this region identi®ed the clone as CaM kinase II d C . The expression of CaM kinase II d in MIN6 b-cells was con®rmed by RT-PCR and by Western blotting. Conclusions Human islets of Langerhans express the d C isoform of CaM kinase II.