Large conductance Ca²⁺ and voltage-activated (BK) channels are pore-forming proteins studded in the membrane of excitable and non-excitable cells. They are ubiquitously expressed and can modify functions as diverse as smooth muscle contraction, neurotransmission and epithelial surface volume. The biophysical properties of BK channels are modulated by regulatory β1-4^1,2 and γ1-4³ subunits in a tissue-specific manner.  We have recently discovered a new family of BK channel regulatory subunits called the LINGO proteins^4-5 which can alter BK plasmalemmal expression to varying degrees, induce inactivation and shift the voltage at which the BK channels activate^4-5.

The focus of this study was to investigate the molecular mechanisms underlying the negative shift in the activation V_1/2 (V_1/2ACT) observed when LINGO_1/2 proteins are co-expressed with BK channels⁴. Recently, cryo-EM studies⁵ have suggested that γ subunits shift BK channel V_1/2ACT by a salt-bridge interaction between D370 in the BK channel and positively-charged residues in the juxta-membrane region of γ subunits. Given that γ and LINGO proteins appear similar in structure, we examined if a similar interaction occurred between BK and LINGO2.

We hypothesised that LINGO2 induced its negative shift in BK V_1/2ACT via an interaction between K574 located intracellularly at the juxta membrane region of LINGO2 and D370 in the RCK1 domain of BK. The effects of disrupting this putative interaction were examined using mutagenesis and electrophysiology.

BK⍺, LINGO2 and eGFP cDNA (100:500:150ng ratio) were transiently co-transfected into HEK cells. Currents were recorded under voltage clamp at 37ËšC from inside out patches from HEK cells that expressed BKα and LINGO2. K⁺ solutions containing 140mM KCl, 10mM glucose, 10mM HEPES, and either 1mM EGTA or HEDTA were used as pipette and internal solutions. Current voltage relationships were determined in patches held at -60mV and stepped from -100mV to +200mV in 20mV increments for 50ms. GV curves were constructed from these and fitted with a Boltzmann equation to determine BK V_1/2ACT. Summary data is presented as mean ± SEM and Student’s unpaired t-test or ANOVA were used to assess statistical significance, as appropriate.

In wildtype BK channels, currents activated in 100 nM Ca²⁺ with a V_1/2ACT of 151±1 mV (n=6) compared to 129±2 mV in BK:LINGO2 channels (n=6, p=0.0085). The V_1/2ACT was unaltered in the BK_D370A mutant (152±2 mV, n=6) compared to wildtype BK channels. In contrast, the V_1/2ACT derived from BK_D370A:LINGO2 currents (163±2 mV) was positively shifted in comparison to BK:LINGO2 (n=6, p=0.0046). Similarly the V_1/2ACT of BK:LINGO2_K574A was shifted positively to  166±2 mV (n=6, p=0.0017) and to 151±2 mV (n=6) in the double BK_D370A:LINGO2_K574A mutant.

In conclusion the shift in V_1/2ACT induced by LINGO2 appears to be due to an interaction between K574 in the LINGO2 tail and D370 in the BK RCK1 domain, possibly due to the formation of a salt bridge between charged residues  in the juxta membrane region of LINGO2 and the N lobe of RCK1 in BK. These data suggest that  γ and LINGO subunits may utilize similar mechanisms to shift activation V1/2 of BK channels.