Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Abstract

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual ␣-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homoand heterotetrameric channels. Only the Kv2 ␣-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.Shaker-related channel subfamilies (Kv1-Kv6 and Kv8 -Kv9) have been identified based on the degree of sequence homology (1). Fully assembled Kv channels are composed of four ␣-subunits arranged around a central pore. Each ␣-subunit consists of six transmembrane segments S1-S6 with a cytoplasmic N and C terminus. The N terminus contains the T1 domain, a tetramerization domain that facilitates the assembly of ␣-subunits into functional channels. The presence of a T1 domain is not absolutely required for channel assembly because subunits without a T1 domain could also assemble into a functional tetramer, although less efficiently (2-4). However, the T1 domain not only promotes but also restricts the formation of possible homo-and heterotetramers by preventing incompatible subunits from assembling (5-7). When four compatible T1 domains assemble, they are arranged with the same 4-fold symmetry as the transmembrane segments, forming a hanging gondola structure (8).The T1 domain contains two subdomains, designated A and B box, which are both highly conserved within the different Kv subfamilies (9). Several residues in both the A and B boxes form the contact interface between neighboring subunits and are commonly assumed to be the key determinants for (subfamily specific) channel tetramer...

Mutational Consequences of Aberrant Ion Channels in Neurological Disorders

“…2a) which has specificity for conducting K ? ions (Bocksteins et al 2009). Auxiliary b-subunit assembles into tetrameric structures and interacts with a-subunit of K v channel which provides a role in modulating channel gating kinetics (Gulbis et al 1999).…”

“…Each a-subunit of voltage-gated potassium channel (K v ) comprised six transmembrane helical segments (S1-S6) with a cytoplasmic N-and C-termini. N-terminus have T1 domain (tetramerization domain) that assembles the a-subunits into functional channel (Zerangue et al 2000;Bocksteins et al 2009), while S4 segment is highly positive and acts as voltage sensor along with the loop between S5-S6 (Yost 1999). Pore region contains the signature sequence GYG (Fig.…”

Immunostaining of Voltage-Gated Ion Channels in Cell Lines and Neurons – Key Concepts and Potential Pitfalls

“…When a trafficking deficiency causes the lack of functional membrane proteins at the PM, FRET could still be observed in the ER while a tetramerization deficiency would also eliminate FRET in the ER. With this approach, it has been demonstrated that the reduced PM expression of mutant Kv2.1 channels was due to a tetramerization deficiency, since the observed reduced current amplitude of those mutant channels was accompanied by a significant reduction of FRET in the ER (Bocksteins et al, 2009). …”

“…In addition to using FRET to investigate whether two proteins of interest only co-localize or actually interact with each other, FRET can also be used to ascertain whether the lack of functional membrane proteins at the PM is due to a trafficking or tetramerization deficiency (Bocksteins et al, 2009). For this purpose, the FRET efficiency in both a region that represents the PM and a region that represents the ER has to be determined.…”

“…In addition to the fact that these chromophores could change the properties of the native protein significantly, it is also possible that those chromophores are introduced into the proteins at positions where the distance between both chromophores is too large to observe FRET. For example, when the interaction between voltage-gated K + subunits is investigated by FRET experiments, the subunits have to be labeled N-terminally and not C-terminally since no FRET could be observed when those subunits are labeled C-terminally although those subunits physically interact with each other in a functional tetrameric Kv channel (Bocksteins et al, 2009;Kobrinsky et al, 2006).…”