Abstract
The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.
Highlights
Large conductance calcium- and voltage-activated potassium (BK)3 channels are uniquely regulated by changes in both transmembrane potential as well as intracellular free calcium levels [1]
We demonstrate that a functionally diverse array of splice variants with a range of calcium and voltage sensitivities, distinct tissue distribution, differential sensitivity to protein phosphorylation, and subcellular localization are generated from a single site of splicing
Multiple Splice Variants Expressed at Site of Splicing C2—Transcript scanning of cDNA generated from 19-day-old whole mouse embryo RNA, using primers designed to amplify across alternative site of splicing C1 and C2 (Fig. 1, a and b), resulted in a family of alternatively spliced transcripts with distinct inserts at site of alternative splicing C1 and C2 (Fig. 1c)
Summary
HEK293 cells were subcultured essentially as described [44, 51]. Cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. For electrophysiological or imaging assays, cells were plated on glass coverslips. Twenty four hours prior to the experiment, cells were washed, and medium was replaced with Dulbecco’s modified Eagle’s medium containing insulin-transferrin-sodium selenite (ITS) serum replacement (Invitrogen). Cells were transiently transfected at 40 – 60% confluence with the respective cDNA using Lipofectamine 2000 (Invitrogen), essentially as described by the manufacturer. Medium was replaced after 24 h, and assays were performed 48 –72 h post-transfection. Stable cell lines were created by selection and maintenance by using 0.8 mg/ml geneticin (Invitrogen). Clones were characterized by agarose gel electrophoresis and sequenced on both strands by automated sequencing (MWG Biotec, Germany)
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