Small conductance Ca2+-activated K+ channels (SK) are widely distributed in both neuronal and non-neuronal tissues. Three genes, SK1, 2, and 3, code for mammalian SK channel subunits (Köhler et al. 1996). Heterologous expression of either the rat SK2 or SK3 genes (rSK2 and rSK3) gives rise to functional homomeric channels, similar in pharmacology to native channels (e.g. Hosseini et al. 2001; Fanger et al. 2001). However, to date attempts to express the rat SK1 (rSK1) gene have not yielded detectable channel activity (Bowden et al. 2001). We have investigated the properties of rSK1 in detail.
Using an antibody selective to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have found that the rSK1 protein is produced but it appears to be trapped in intracellular compartments. Similar results have been reported for known silent subunits (Post et al. 1996).
To test the idea that rSK1 is also a silent subunit we co-expressed rSK1 with rSK2 and found a 67 ± 22 % (mean ± S.E.M.) increase in Ca2+-activated K+ conductance compared with cells transfected with rSK2 alone. Moreover, in co-transfected cells the sensitivity of the channels to block by apamin and UCL 1848 was substantially reduced (IC50 values for apamin and UCL 1848 were 95 ± 8 pM (mean ± S.D.) and 110 ± 26 pM, respectively, for inhibition of rSK2 alone compared with 1.4 ± 0.3 nM and 2.9 ± 0.3 nM for inhibition of rSK1/rSK2 currents). These findings indicate that rSK1 is able to reach the cell surface as an SK1/SK2 heteromer.
In contrast, co-transfection of rSK1 with rSK3 caused an 84 ± 4 % reduction in current level compared with cells expressing SK3 alone. By using a TEA-sensitive mutant of SK3 (SK3V515F), we were able to show that the TEA sensitivity of this residual current is similar to that of cells expressing SK3V515F alone, suggesting that there is little or no contribution from rSK1 and that most of the channels are probably homomeric rSK3 assemblies.
In conclusion our data support the idea that rSK1 can interact with both rSK2 and rSK3. The different interactions between rSK1 and the other subunits suggest that trafficking of the channels is influenced by subunit composition. Further, rSK1 appears to act as a silent subunit.
This work was supported by the Wellcome Trust.