Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon “SRKR”

Abstract

BK Ca2+-activated K+ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca2+-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK0 and BKSRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK0 and BKSRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK0, BKSRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca2+ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca2+-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BKSRKR channels were significantly reduced compared with BK0, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.

Calcium activated K+ channels in the electroreceptor of the skate confirmed by cloning. Details of subunits and splicing

“…5 (left) also shows the 6-7 amino acids preceding the site 3 boundary marker, which are similar in the chick cochlea, skate ampullae and skate embryos. The four amino acid insert, SRKR, which has been found at site 3 in rat and human B K channels [39] was not found in the assembled skate embryo channel, or in the ampulla. The SRKR insert plays a role in circadian rhythm in the rat and other species.…”

Shaw and Shal voltage‐gated potassium channels mediate circadian changes in Drosophila clock neuron excitability

“…Phosphorylation may also be an important post‐translational means of circadian regulation of ion channel activity, with mammalian slo or BK being under phosphorylation control in the SCN (Shelley et al . ). Circadian phosphorylation by clock kinases maybe a more general bi‐directional link between the molecular clock and membrane ion channels that regulate the rhythmic firing of clock neurons (Ko et al .…”

“…Null mutants sleep a third as long as normal flies resulting in flies with significant reductions in lifespan (Cirelli et al 2005;Bushey et al 2007;Kempf et al 2019). Phosphorylation may also be an important post-translational means of circadian regulation of ion channel activity, with mammalian slo or BK being under phosphorylation control in the SCN (Shelley et al 2013). Circadian phosphorylation by clock kinases maybe a more general bi-directional link between the molecular clock and membrane ion channels that regulate the rhythmic firing of clock neurons (Ko et al 2009;Allada & Chung 2010;Allen et al 2017).…”

Nuclear localization of calcium-activated BK channels in skate ampullary electroreceptors

“…Functional Kv10.1 channels localized to the inner nuclear membrane together with Lamin A/C subsequent to exposure to the extracellular milieu [33]. BK channels are regulated by complex alternative splicing, post-translational modifications and protein-protein interactions that together influence subcellular localization and function [27,[34][35][36][37][38][39][40][41][42][43][44]. Alternative splicing at the C-terminus that incorporates amino acids -DEC targets BK channels to mitochondria [45].…”