[Abstract Title]. - Society for Neuroscience

a shRNA knockdown approach to selectively reduced TRPC1 expression levels by transient
transfection of multiple TRPC1 shRNA plasmids that target different sequences of the gene into
D54MG cells. This led to a significant reduction in TRPC1 protein expression as determined by
Western blot and significantly reduced TRPC1 mediated SKF96365 sensitive currents as
determined by patch-clamp recordings. Furthermore, FURA-2 imaging studies show that store
operated calcium entry was impaired in cells that have been transfected with shRNA plasmids.
Finally, transfection with shRNA also slowed glioma proliferation and a proportion of
transfected cells became large and multinucleated as we had previously observed with
pharmacological inhibition of TRPC1 channels. These studies suggest that TRPC1 constitutes a
molecularly defined component of the store-operated Ca 2+ entry pathway required to allow
glioma cells to undergo normal cell divisions. (supported by NIH 5RO1 NS031234 & 2RO1
NS036692).
Disclosures: V.C. Bomben, None; H.W. Sontheimer, None.
Poster
235. Ion Channels in Disease I
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 235.13/C59
Topic: B.04.d. Ion channels and disease
Support: NIH Grant EY014852 and GM60448
Title: Regulation of bestrophin Cl channels by calcium and phosphorylation: role of the Cterminus
Authors: *Q. XIAO 1 , K. YU 1 , A. PRUSSIA 2 , Y. CUI 1 , H. C. HARTZELL 1 ;
1 Cell Biol., 2 Dept. of Chem., emory university, Atlanta, GA
Abstract: Bestrophins, a new family of chloride channels, are activated by Ca. However, the
structures and mechanisms responsible for Ca sensitivity remain unknown. hBest1 contains a
region of acidic amino acids that has been proposed as a potential Ca binding site. Of the 35
mutants we made in this region, all but two were non-functional. Therefore, we searched for
other Ca binding domains and identified a pair of motifs in the C-terminus that resemble EFhands.
Using a homology model built on the crystal structure of calmodulin, the first EF hand of
hBest1 was predicted to bind Ca with higher affinity than the third EF hand of calmodulin. We
made point mutations in the first EF hand of hBest1 to test whether this region was involved in
activation of the channel by Ca. The D312G mutant in the putative Ca binding loop (312-323)
reduced Ca affinity by 20-fold, and also abolished Ca-dependent rundown. In contrast, mutations