Dipeptidyl‐peptidase‐like proteins cast in a new role: enabling scorpion toxin block of A‐type K+ channels

Subthreshold-operating A-type K+ currents exhibiting rapid inactivation are major regulators of somatodendritc excitability and plasticity in the nervous system (Jerng et al. 2004; Maffie & Rudy, 2008). There is also compelling evidence indicating that the Kv4.2 and Kv4.3 pore-forming subunits are significant molecular correlates of these currents (Maffie & Rudy, 2008). Although these subunits can form functional homo- and heterotetrameric A-type K+ channels, they need essential partners to recapitulate their native phenotype and perform their functions (Maffie & Rudy, 2008). Two distinct ancillary subunits are confirmed components of the Kv4 channel complex in neurones, namely the K+ channel-interacting proteins (KChIPs) and dipeptidyl-peptidase-like proteins (DPPs) encoded by various genes (KChIP1–4, DPP6 and DPP10), which generate diverse splice variants. Current structural models of the ternary Kv4 channel complex suggest a dodecameric structure, in which there is one KChIP and one DPP for every Kv4.2/Kv4.3 subunit in the pore-forming tetrameric channel (Covarrubias et al. 2008). The DPPs, in particular, affect almost every aspect in the life of the Kv4 channel complex. They increase surface expression and unitary conductance, and promote activation and inactivation gating. Dipeptidyl-peptidase-like proteins are single-pass membrane proteins with an intracellular N-terminal region and a large extracellular C-terminal region constituting ∼90% of the mass of the protein. While the N-terminal and membrane-spanning regions are mainly responsible for the gating effects, the roles of the C-terminal region are largely unknown. Recent work investigated CA1 hippocampal neurons from wild-type and DPP6 knockout (DPP6 KO) mice to determine that DPP6 establishes the somatodendritic gradient of the A-type K+ current and thereby helps regulate backpropagating action potentials and long-term potentiation (Sun et al. 2011). Whether or not the DPP6 C-terminal region influences subcellular localization is unknown. Also, despite the wealth of knowledge about the biophysical, cellular and physiological roles of DPPs, not much is known about their possible influence on the pharmacology of the ternary Kv4 channel complex. Given the extracellular topology of DPPs and their putative direct interactions with pore-forming subunits, such a role is likely. In order to investigate the functions of somatodendritic A-type K+ channels in short-term conditions, specific high-affinity blockers of neuronal Kv4 ternary complexes would be useful for both in vivo and in vitro studies. Knockdown and knockout approaches are often confounded by long-term compensation and remodelling of native conductances. In this issue of the The Journal of Physiology, Maffie et al. (2013) report compelling evidence for the new role of DPPs as determinants of the potent block of neuronal A-type K+ channels by the peptide toxin AmmTx3, a member of the α-KTX15 family of scorpion toxins. At the same time, these authors solved the apparent discrepancy between the scorpion toxin sensitivity of native and heterologously expressed Kv4.2/Kv4.3 channels, which had hindered the experimental applications of the aforementioned toxins. While the native neuronal channels are blocked by submicromolar concentrations of AmmTx3, the recombinant counterparts are only modestly inhibited by similar concentrations. At the centre of this study is the investigation of the granule neuron. A-type K+ current in cerebellar slices from wild-type and DPP6 KO mice. Clearly, 500 nm AmmTx3 nearly eliminates the A-type component of the total current. In contrast, demonstrating that high AmmTx3 sensitivity depends on the expression of DPP6, almost 90% of the A-type K+ current from the DPP6 KO mouse is resistant to the same concentration of the toxin. The authors also show that AmmTx3 is not a gating modifier and that it is most likely to act as a high-affinity external pore blocker (IC50≍ 100 nm), resembling analogous scorpion toxins acting on related Ca2+- and voltage-gated K+ channels. This important conclusion will facilitate the interpretation of results in future applications. Then, in order to show that DPP6 is definitely the culprit behind the enhanced AmmTx3 sensitivity of the neuronal A-type K+ current, the authors also reconstituted low- and high-affinity block in heterologously expressed Kv4.2 channels. Essentially, only when DPP6 was present in binary (Kv4.2+DPP6 vs. Kv4.2+KChIP1) and ternary complexes (Kv4.2+DPP6+KChIP1) did the expressed currents exhibit AmmTx3 block comparable to that observed in cerebellar granule neurons. Moreover, heterologous coexpression of Kv4.3 and DPP10 also induced A-type K+ currents exhibiting high scorpion toxin sensitivity, indicating that all known DPPs that form Kv4 channel complexes are capable of enhancing the apparent affinity of AmmTx3. How do DPPs accomplish this surprising feat? In the light of the available structural information (Covarrubias et al., 2008; Maffie & Rudy, 2008), one could imagine a role for the intriguing C-terminal region of DPPs. Like large balloons tethered to the membrane, the massive DPP dimer of dimers might hover immediately above the external vestibule of the pore but, rather than hindering toxin binding, they enhance it. Perhaps, the membrane-facing side of the extracellular C-terminal region and juxtamembrane linkers sculpt the external vestibule of the pore in Kv4.2/Kv4.3 channels to promote stronger toxin binding. Direct structural approaches will be necessary to solve this puzzling question conclusively. Additionally, it would be important to determine whether AmmTx3 also blocks native and recombinant Kv4.1 channels in a DPP-dependent manner. The Kv4.1 and Kv4.3 subunits are expressed in sensory neurons, but the subunit composition of the Kv4 channel complex in this cell type remains unknown (Phuket & Covarrubias, 2009). At any rate, the emergence of new tantalizing questions with broad implications signifies progress. Demonstrating specific DPP-dependent block of somatodendritic A-type K+ channels by AmmTx3 will undoubtedly stimulate further research on the functions, structure and pharmacology of neuronal Kv4 channel complexes and their possible roles in disease states, such as epilepsy, chronic pain, neurodegenerative disorders and autism.