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.

Hill number (1.3) for this HENA action were undistinguishable from those of LCA, HENA's efficacy was significantly larger than that of LCA, with channel steady-state activity in presence of 150 mM HENA increasing to 200% of pre-drug values. Remarkably, HENA failed to activate recombinant, b2-, b3-, or b4-containing BK channels while activating native BK channels in rat cerebrovascular myocytes (EC 50 =46 mM). Therefore, HENA selectively targets b1-containing BK channels. Furthermore, HENA failed to activate cbv1þb1T169A channels, suggesting it acts via a BK b1 cholane-sensing site (Bukiya et al., 2008). HENA (3-45 mM) dilated pressurized cerebral arteries of rat and C57BL/6 mouse. Consistent with its higher efficacy on channel activity, HENA-induced dilation was significantly larger than that of LCA. As expected, genetic ablation of BK b1 suppressed HENA-induced vasodilation. Finally, closed cranial window data from anesthesized rats demonstrated that intracarotid infusion of HENA significantly dilated pial arterioles. This action was sustained in presence of 4-aminopyridine but totally prevented by paxilline, underscoring BK-mediation of HENA-induced in vivo vasodilation. This study identifies the first selective non-steroid activator of b1-containing BKs and effective cerebral artery dilator. Supported by R01-HL104631; R37-AA011560 (AMD).