Structural Determinants of M-Type KCNQ (K_v7) K⁺Channel Assembly

Abstract

The ability of KCNQ (K v 7) channels to form hetero-oligomers is of high physiological importance, because heteromers of KCNQ3 with KCNQ2 or KCNQ5 underlie the neuronal M-current, which modulates neuronal excitability. In KCNQ channels, we recently identified a C-terminal subunit interaction (si) domain that determines their subunit-specific assembly. Within this si domain, there are two motifs that comprise ϳ30 amino acid residues each and that exhibit a high probability for coiled-coil formation. Transfer of the first or the second coiled-coil (TCC) domain from KCNQ3 into the KCNQ1 scaffold resulted in chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3, both of which coimmunoprecipitated with KCNQ2. However, only KCNQ1(TCC2)Q3 enhanced KCNQ2 currents and surface expression or exerted a strong dominant-negative effect on KCNQ2. Deletion of TCC2 within KCNQ2 yielded functional homomeric channels but prevented the current augmentation measured after coexpression of KCNQ2 and KCNQ3. In contrast, deleting TCC1 within KCNQ2 did not give functional homomeric KCNQ2 or heteromeric KCNQ2/KCNQ3 channels. Mutations that disrupted the predicted coiled-coil structure of TCC1 in KCNQ2 or KCNQ3 abolished channel activity after expressing these constructs singly or in combination, whereas helix-breaking mutations in TCC2 of KCNQ2 gave functional homomeric channels but prevented the heteromerization with KCNQ3. In contrast, KCNQ3 carrying a coiled-coil disrupting mutation in TCC2 hetero-oligomerized with KCNQ2.Our data suggest that the TCC1 domains of KCNQ2 and KCNQ3 are required to form functional homomeric as well as heteromeric channels, whereas both TCC2 domains facilitate an efficient transport of heteromeric KCNQ2/KCNQ3 channels to the plasma membrane.

A Pore Residue of the KCNQ3 Potassium M-Channel Subunit Controls Surface Expression

“…The impact of the C terminus was calculated assuming the previous figure for ER retention. The resulting figure (3.6) is similar to the reported impact on KCNQ3 surface expression of a cytosolic fragment of helix D from KCNQ2 (Schwake et al, 2006).…”

“…This is not, however, the only means of promoting KCNQ3 exit. It is well established that heteromerization of the C terminus plays a role on the surface expression increase after KCNQ2 and KCNQ3 assembly (Maljevic et al, 2003;Schwake et al, 2006;Nakajo and Kubo, 2008). Previously we showed that a chimeric subunit in which the C terminus of KCNQ3 was substituted for that of KCNQ2 also promoted the efficient traffic of KCNQ3 (Etxeberria et al, 2004), suggesting that a complex in which all subunits have alanine within the pore inner vestibule can still escape retention.…”

“…On the other hand, assembly of the C-terminal region with helix D of KCNQ2 results in an additional 3.6-fold increase in plasma membrane expression (Schwake et al, 2006). Thus, when KCNQ3 assembles with KCNQ2 the final increase in surface would be 22-fold, i.e., the product of the contribution of the pore and assembly domains (see Fig.…”

“…This amino acid plays a critical role in inhibiting the function of KCNQ3 homomeric channels (Etxeberria et al, 2004), but the mechanism is unknown. It is generally thought that KCNQ2/3 surface expression is governed by the intracellular C-terminal region, and that the pore region does not have any significant role in trafficking (Maljevic et al, 2003;Schwake et al, 2003Schwake et al, , 2006Etxeberria et al, 2004;Nakajo and Kubo, 2008;Zaika et al, 2008). This presumption and the observation that KCNQ3-A315T is functional have led to the suggestion that KCNQ3 may be present at the plasma membrane in an electrically silent conformation (Etxeberria et al, 2004;Zaika et al, 2008).…”

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation

“…In contrast to the obligatory role of helices ABC for Kv7.2 channel function, helix D is dispensable (Schwake et al, 2006;Nakajo and Kubo, 2008). The helix D coiled-coil bundle has been largely considered as a passive stitch, with little active role other than to convey stability to the tetramer and specificity during the formation of heteromeric assemblies (Schmitt et al, 2000;Jenke et al, 2003;Maljevic et al, 2003;Schwake et al, 2006;Howard et al, 2007).…”