Assembly of M-type KCNQ channnels

128

165

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOHterminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The firstcoiled-coilcomplexisdimericandundergoesconcentrationdependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 Å crystallographic data. Both coiledcoils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.

Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 92%

See 1 more Smart Citation

The KCNQ1 (Kv7.1) COOH Terminus, a Multitiered Scaffold for Subunit Assembly and Protein Interaction

Wiener

Haitin

Shamgar

et al. 2008

128

165

“…On the one hand, the KCNQ3 subunit can bind CaM, such that there are at least two intact CaMbinding sites available in the heterotetramers. On the other hand, heteromerization causes an increase in surface expression that is dependent on the assembly domain formed by helices C and D (3,18). It is important to note that small changes in adopting the active configuration translate into a large reduction in the number of functional channels at the plasma mem- brane.…”

Section: Discussionmentioning

confidence: 99%

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

Alaimo

Gómez-Posada

Aivar

et al. 2009

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K ؉ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca 2؉ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca 2؉ sensor, conferring Ca 2؉ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

“…Although co-expression data in oocytes suggest that KCNQ3 and KCNQ4 might coassemble (8), that conclusion still seems tentative. On the other hand, KCNQ2 has been clearly shown to not assemble with any other type subunit besides KCNQ3 (3,8), and KCNQ1, although robustly assembling with the family of KCNE ␤-subunits to form a variety of heteromeric channels with different properties, does not assemble with any of the other KCNQ2-5 subunits (3,8,16,23,24). The possibility that KCNQ4 and KCNQ5 might co-assemble to form functional channels is as yet unexplored.…”

Section: M-type Kmentioning

confidence: 99%

Homomeric and Heteromeric Assembly of KCNQ (Kv7) K+ Channels Assayed by Total Internal Reflection Fluorescence/Fluorescence Resonance Energy Transfer and Patch Clamp Analysis

Bal

Zhang

Zaika

et al. 2008

M-type K؉ channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent proteinand yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.The KCNQ (Kv7) family of voltage-gated channels underlie a number of important K ϩ currents throughout the body, including the M current of neurons produced by the homomeric and heteromeric assembly of KCNQ2, KCNQ3, and KCNQ5 (1-7). KCNQ4 makes homomeric K ϩ channels important for K ϩ transport primarily in the inner ear (8, 9), whereas KCNQ1 assembles with KCNE ␤-subunits to form several important currents in heart, ear, and epithelia (10, 11). The understanding of the different subunit compositions of KCNQ channels is important because such compositions affect the properties of the channels. Thus, expression of KCNQ2 and KCNQ3 individually yields only small whole-cell currents, whereas their coexpression yields heteromeric currents 10-fold larger (5, 12-16). Homomeric KCNQ2, -4, and -5 channels are sensitive to Ca 2ϩ /calmodulin, whereas KCNQ1 and KCNQ3 are not (17). Finally KCNQ1 and KCNQ2 subunits in particular have been shown to be modulated by phosphorylation by protein kinases A and C, respectively, via complexes assembled by the scaffolding proteins yotiao and AKAP79/150 (18 -21). Therefore, it is expected that the inclusion of these different subunits in the channel tetramer should confer specific properties characteristic of each...