Neuronal fast activating and meningeal silent modulatory BK channel splice variants cloned from rat

Abstract

The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned from rat brain or cerebral vascular/meningeal tissues, of which at least three variants were previously uncharacterized (X1, X2(92), and X2(188)). An additional variant was identified by polymerase chain reaction but not cloned. Expression in Xenopus oocytes showed that the brain-specific X1 variant displays reduced current, faster activation, and less voltage sensitivity than the insert-less Zero variant. Other cloned variants Strex and Slo27,3 showed slower activation than Zero. The X1 variant contains sequence inserts in the S1-S2 extracellular loop (8 aa), between intracellular domains RCK1 and RCK2 (4 aa at SS1) and upstream of the calcium "bowl" (27 aa at SS4). Two other truncated variants, X2(92) and X2(188), lacking the intracellular C-terminal (stop downstream of S6), were cloned from cerebral vascular/meningeal tissue. They appear non-functional as no current expression was observed, but the X2(92) appeared to slow the activation of the Zero variant when co-expressed. Positive protein expression of X2(92) was observed in oocytes by immunoblotting and fluorescence using a yellow fluorescent protein-tagged construct. The functional characteristics of the X1 variant may be important for a subpopulation of BK channels in the brain, while the "silent" truncated variants X2(92) and X2(188) may play a role as modulators of other BK channel variants in a way similar to well known beta subunits.

Identification and quantification of full‐length BK channel variants in the developing mouse cochlea

“…Moreover, functional evidence suggests that a truncated form of BK, which lacks the entire C‐terminus, still shows wild‐type Ca 2+ sensitivity (Piskorowski and Aldrich,2002). A recent study, however, suggests a second alternative in that truncated N‐BK modulates BK variants, such as ZERO, by slowing activation and increasing membrane expression, while showing no activity on its own (Poulsen et al,2011). These reported variants extend 92 and 188 aa downstream of S6.…”

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

“…In order to faithfully measure the localization of endogenous BK channels, we generated a gene-targeted mouse in which hemagglutinin (HA) and FAP tag were inserted into at the endogenous BKα locus at exon 1, which is incorporated into all known functional BKα ( Erxleben, 2002 ; Poulsen et al, 2011 ; Sakai et al, 2011 ). FAP-BKα channels exhibit similar voltage and calcium responses as untagged BKα.…”

Large Conductance Ca2+-Activated K+ Channel (BKCa) α-Subunit Splice Variants in Resistance Arteries from Rat Cerebral and Skeletal Muscle Vasculature

“…Up to ten alternative splicing sites have been identified for the vertebrate BK Ca α-subunit [11]. Most variation occurs in the intracellular C-terminal part in the linker region between domains RCK1 and RCK2 and upstream of the “calcium bowl” [12]. Alternative splicing can modify the functional properties of BK Ca channels, including Ca 2+ and voltage sensitivity, cell surface expression, and regulation by diverse intracellular signaling pathways.…”