Molecular Components of Large Conductance Calcium-Activated Potassium (BK) Channels in Mouse Pituitary Corticotropes

Abstract

Large-conductance calcium- and voltage- activated potassium (BK) channels play a fundamental role in the signaling pathways regulating mouse anterior pituitary corticotrope function. Here we describe the cloning and functional characterization of the components of mouse corticotrope BK channels. RT-PCR cloning and splice variant analysis of mouse AtT20 D16:16 corticotropes revealed robust expression of mslo transcripts encoding pore-forming alpha-subunits containing the mouse homolog of the 59-amino acid STREX-1 exon at splice site 2. RT-PCR and functional analysis, using the triterpenoid glycoside, DHS-1, revealed that native corticotrope BK channels are not functionally coupled to beta-subunits in vivo. Functional expression of the STREX-1 containing alpha-subunit in HEK 293 cells resulted in BK channels with calcium sensitivity, single-channel conductance, and inhibition by protein kinase A identical to that of native mouse corticotrope BK channels. This report represents the first corticotrope ion channel to be characterized at the molecular level and demonstrates that mouse corticotrope BK channels are composed of alpha-subunits expressing the mouse STREX-1 exon.

Alternative Splicing Switches Potassium Channel Sensitivity to Protein Phosphorylation

“…GST fusion proteins of the STREX-1 splice insert were constructed in pGEX-5x-1 by polymerase chain reaction and recombinant protein purified and assayed in a PKA phosphorylation reaction under standard conditions. Coimmunoprecipitation of channel variants with the catalytic subunit of PKA (PKAc) was performed essentially as described previously (24).Electrophysiology-All experiments were performed in the inside-out configuration of the patch clamp technique at room temperature (20 -24°C) using physiological potassium gradients essentially as described previously (22). The pipette solution (extracellular) contained (in mM): 140 NaCl, 5 KCl, 0…”

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

“…To further address the requirement for a functional LZ1 domain for PKA action, the third and fourth d position LZ1 leucine residues (Leu-530 and Leu-537) in the HA-tagged STREX channel were mutated to alanine and expressed in HEK293 cells. Although the halfmaximal voltage for activation of STREX channels is shifted to more negative potentials than for ZERO channels (25), the LZ1 mutant STREX channels (STREX L530A/L537A ) expressed in HEK293 cells displayed no significant shift in their half-maximal activation voltage compared with wild type STREX channels under the assay conditions used. In the presence of 0.2 M free calcium and Mg-ATP, the respective V 0.5 was 47 Ϯ 8 mV, n ϭ 4 (STREX) and 52 Ϯ 5 mV, n ϭ 4 (STREX L530A/L537A ).…”

“…To assay for regulation of BK channel splice variants by endogenous PKA we applied cAMP to the intracellular face of isolated inside-out patches from HEK293 cells to activate PKA closely associated with the channel as previously reported (17). The mouse ZERO variant of BK is activated by PKA closely associated with the channel and dependent upon a C-terminal serine residue (Ser-899) conserved in all mammalian BK channel splice variants (17,25). C-terminal HA-tagged ZERO channels were stimulated upon application of cAMP to the intracellular face of inside-out patches from HEK293 cells only in the presence of Mg-ATP (48.6 Ϯ 8.0%, n ϭ 8, Fig.…”

“…STREX channels are identical to ZERO apart from inclusion of a 59-amino acid insert (17,25). The mouse STREX splice variant is potently inhibited by PKA closely associated with the channel complex and dependent upon a conserved serine residue within the STREX insert (17).…”

“…Variant and Mutant cDNA Constructs-The cloning and sub-cloning of the mouse BK channel splice variants ZERO and STREX into the mammalian expression vector pcDNA3 or pcDNA3.1ϩ (Invitrogen) have been described previously (17,25). A C-terminal hemagglutinin (HA) tag was introduced into each channel construct by replacing the normal stop codon with a sequence encoding the HA tag from a mouse BK channel construct kindly provided by Dr. Yi Zhou and Prof. Irwin B Levitan.…”