Distal End of Carboxyl Terminus Is Not Essential for the Assembly of Rat Eag1 Potassium Channels

Abstract

The assembly of four pore-forming ␣-subunits into tetramers is a prerequisite for the formation of functional K ؉ channels. A short carboxyl assembly domain (CAD) in the distal end of the cytoplasmic carboxyl terminus has been implicated in the assembly of Eag ␣-subunits, a subfamily of the ether-à-go-go K ؉ channel family. The precise role of CAD in the formation of Eag tetrameric channels, however, remains unclear. Moreover, it has not been determined whether other protein regions also contribute to the assembly of Eag subunits. We addressed these questions by studying the biophysical properties of a series of different rat Eag1 (rEag1) truncation mutants. Two truncation mutants without CAD (K848X and W823X) yielded functional phenotypes similar to those for wild-type (WT) rEag1 channels. Furthermore, nonfunctional rEag1 truncation mutants lacking the distal region of the carboxyl terminus displayed substantial dominant-negative effects on the functional expression of WT as well as K848X and W823X channels. Our co-immunoprecipitation studies further revealed that truncation mutants containing no CAD indeed displayed significant association with rEag1-WT subunits. Finally, surface biotinylation and protein glycosylation analyses demonstrated that progressive truncations of the carboxyl terminus resulted in aggravating disruptions of membrane trafficking and glycosylation of rEag1 proteins. Overall, our data suggest that the distal carboxyl terminus, including CAD, is dispensable for the assembly of rEag1 K ؉ channels but may instead be essential for ensuring proper protein biosynthesis. We propose that the S6 segment and the proximal carboxyl terminus may constitute the principal subunit recognition site for the assembly of rEag1 channels.

The Enigmatic Cytoplasmic Regions of KCNH Channels

“…This effect was largely erased when the PAS domain was also truncated, suggesting that the two regions influence each other. In rat EAG and hERG channels, a 40–50 amino acid stretch close to the C-terminus predicted to form a coiled-coil was implicated in subunit oligomerization 68; 69 , but more recently this conclusion has been challenged 70 .…”

Voltage-Gated Potassium Channels (Kv10–Kv12)☆

“…This cytoplasmic assembly likely contributes both to the stability of the tetrameric channel assembly as well as contributing to the fine-tuning of the voltage-dependent gating of these channels (see below). Whilst multiple cytoplasmic domains, as well as the pore domain, contribute to assembly of the tetrameric channel complex it is likely that the 6th TM domain and proximal c-terminal linker (that joins the 6th TM domain to the cNBH domain) forms the core tetramerization motif in the KCNH family of channels (Akhavan et al, 2003;Chen et al, 2011a). This contrasts with the situation for other VGK channels where the principle tetramerization domain is located in the cytoplasmic N-terminus (Kreusch et al, 1998;Li et al, 1992).…”

The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

“…The truncation mutants rEag1-K848X and hErg-R1032X produced functional K ϩ channels with biophysical properties similar to those of respective WT (16) (data not shown). By contrast, neither rEag1-N673X nor hErg-N861X generated significant K ϩ currents (16,17). Despite the absence of CAD/TCC, all of the four truncation mutants preserved their native subfamily-specific assembly properties (Fig.…”

“…For Kv1 channels, intersubunit associations via the T1 domain are considered the initial step of tetramerization during the biogenesis and may not be required for subsequent steps in channel assembly (27,36). It is therefore likely that once the initial subfamily recognition process is accomplished, the ensuing protein maturation process leading to the tetrameric organization of a functional ether-à-go-go K ϩ channel may instead involve subunit assembly via other protein domains, such as the S6-C-linker region (16).…”

“…Most importantly, is CAD/TCC the sole and/or principal assembly and recognition domain for Eag and Erg? Previous truncation analyses from our laboratory indicate that the CAD is not required for the assembly of Eag subunits (16). FIGURE 1.…”