Phosphorylation of a Constitutive Serine (S642) in BK Channel Variants Containing the Alternate Exon ‘SRKR’ Alters Current Properties

Abstract

Kcnma1, the gene encoding the pore forming subunit of the BK Ca2+-activated K+ channel, undergoes extensive alternative splicing across tissues, tuning BK current properties. Phosphorylation and β subunit association further contribute to diverse BK current properties, but the interplay of these mechanisms have not been well-investigated in BK variants cloned from native tissues. We used RT-PCR to identify two Kcnma1 splice variants in the mouse suprachiasmatic nucleus (BK0 and BKSRKR). Both variants contain an alternate translation start (MANG) compared to the canonical start (MDAL), no insert at splice site 2 (STREX), an insert at site 3 (near the Ca2+ bowl), as well as an alternate C-terminus (VYR). However, they differ at splice site 1, containing a four amino acid SRKR alternate exon located near the end of the RCK1 domain. Voltage-clamp recordings from the variants expressed in HEK293 cells revealed that BKSRKR channels produced BK currents with distinct properties from the insertless BK0 variant, with significantly right-shifted G-Vs, slower activation, and faster deactivation kinetics at 0, 1, and 100 μM Ca2+. Co-expression of the β4 subunit enhanced these differences in a Ca2+-dependent manner. Inclusion of SRKR resulted in two predicted phosphorylation sites in BKSRKR, S642 (a constitutive residue present in both variants) and S644 (within the SRKR exon). Alkaline phosphatase treatment and mutations at both positions revealed that phosphorylation of S642 is both necessary and sufficient to produce the observed differences between BK0 and BKSRKR current properties. These data demonstrate that phosphorylation of S642 underlies the differences in current properties between BKSRKR and BK0, and suggests the alternate exon SRKR serves to enhance S642 phosphorylation in native tissues.