BK channels, because of their large single-channel conductance and co-activation by depolarization and intracellular Ca++, play an important role in regulating neuronal firing. BK channels disproportionately contribute to neuronal whole-cell K+ channel currents, where they can carry more than half the total voltage-gated K+ current. We have shown that expression of cell-surface BK channels is controlled by the presence of the brain-specific β4 subunit. Using a novel FAP to track the location of tagged BK channels at the cell surface and cytoplasm, we find that coexpression of β4 significantly reduces BKα channel protein at the plasma membrane, a function that is dependent upon a C-terminal ER retention/retrieval motif.
In CA3 neurons, which exhibit the highest levels of β4 expression compared to almost any neurons within the rodent CNS, pharmacologically-isolated whole-cell BK channel currents display none of the expected characteristics for BKα+ β4 channels, suggesting that these channels may not be present at the cell surface. Furthermore, genetic ablation of the β4 subunit was sufficient to significantly increase whole-cell BK channel currents in knock-out animals. Thus, we propose that an important function of the CNS-specific BK channel accessory subunit β4 in CA3 neurons is control of cell-surface trafficking of the BK channel complex.
These studies employed a FAP tag to track the location of BK channels in living cells. This methodology has significant advantages compared to more standard techniques, such as GFP protein tags, where the surface fluorescence signal can be overwhelmed by the much larger intracellular stores of channel protein. Although the GFP-based Phluorin 
, a fluorescence protein tag whose signal is quenched in acidic cell compartments, has been useful for studying vesicle fusion 
, the nearly neutral pH of the ER has been associated with breakthrough fluorescence that complicates analysis. Advantages of the FAP system are use of membrane permeable and impermeable dyes, high signal-to-noise due to low fluorescence of unbound dye, and the potential to carry out real-time imaging experiments. Since unbound dye has essentially no fluorescence, a specific signal is generated without washing off excess dye, a property that will enable imaging in more complex tissue environments. Additionally, because the fluorescence signal from a single- dye-FAP interaction can be so bright (5 to 20-fold brighter than GFP), this technology may be particularly well-suited to studying the localization and dynamics of individual molecules, such as single BK channels at the plasma membrane.
Much experimental effort has gone into understanding the biophysical consequences of β4 on BK channel function, with little attention to how this auxiliary subunit can control channel localization. Using live-cell imaging and immunolocalization in heterologous cells, as well as electrophysiological measurements in CA3 neurons that express high levels of β4, we find that β4 expression is associated with a dramatic reduction of BK channels at the cell surface. In addition, we find that genetic ablation of β4 is sufficient to significantly enhance whole-cell BK channel currents. This may explain the paradoxically broad expression of β4 in the CNS ( and 
) despite iberiotoxin-pharmocology indicating that BK channels lack β4 in many neurons 
. Taken together, our results suggest that the effect of β4 on channel function may be much more indirect than previously imagined.
Nevertheless, a few studies report the presence of iberiotoxin-insensitive BK currents in the CNS, specifically in posterior pituitary nerve terminals and dentate gyrus granule neuronal soma and their mossy fiber terminals 
. Further, the β4 subunit was found to promote surface expression of the related slo3 channel in Xenopus oocytes 
. How can the present findings be resolved with this? Our comparison of iberiotoxin and paxilline-sensitive currents in dentate gyrus neurons indicates that in some neurons, β4 is not sufficient to reduce cell-surface channel expression. Thus, there may be additional factors, including activation of signaling pathways, which can regulate channel localization in a cell-type specific manner.
Some studies have also suggested that the β4 subunit may regulate subcellular distribution of the BK channels into axonal or dendritic compartments 
. Because of the limitations of voltage clamp in neurons (i.e. poor voltage control in the distal processes of the cell), such β4-containing channels might be hard to detect in CA3 neurons. It is also possible that BK channels can be redistributed to the plasma membrane under some circumstances, for example, following mechanical dissociation of cells prior to analysis 
, or during periods of high firing. Redistribution could occur over very short time intervals (100 s of ms), during firing bursts, or might be regulated over the course of many minutes. Such regulation has been observed for Kv
2.1 type K+
. Our finding that β4 expression is associated with the regulation of surface levels of BK channels suggests a new mechanism for dynamic control of channel activity.
A caveat of the present study is that direct association of the BKα subunit with β4 was not directly demonstrated in transfected cells. However, the rescue of surface expression with co-transfection of the β4 mutant construct suggests that there is some interaction between the two proteins. Furthermore, our preliminary recordings from transfected HEK-293 cells indicate that the increase in surface expression observed in BKα+β4-Ala expressing cells is linked to iberiotoxin-resistant whole-cell currents. Further studies will be required to unambiguously demonstrate the interaction of BKα and β4 in our experimental preparation.
Enhanced BK channel currents have been linked to neuronal hyperexcitability and epilepsy in cortical neurons 
. Indeed, BK channel antagonists can reduce bursting in abnormally active tissue 
and have a profound anticonvulsant effect in vivo 
. The large contribution of BK channels to the total K+
channel current in CA3 neurons suggests that these channels may play a particularly important role in controlling the excitability of these cells.
The data presented here characterize the detailed molecular mechanisms that control cellular BK channel trafficking. We have identified a new role for the BK β4 channel subunit – ER retention – as well as the specific amino acid residues that are necessary to direct this function. Additionally, we have shown that BK channels in CA3 neurons are not associated with the pharmacological and biophysical hallmarks of β4-containing channels as described in previous studies using heterologous cells, and that the absence of β4 is sufficient to significantly enhance whole-cell BK channel current. Because these studies were carried out in CNS neurons in acute brain slices, these findings may be particularly relevant to channel function in vivo. The dynamic regulation of endogenous BK channel localization in neurons is an exciting avenue for future investigations.