INTRACELLULAR REGIONS OF THE EAG POTASSIUM CHANNEL PLAY A CRITICAL ROLE IN GENERATION OF VOLTAGE-DEPENDENT CURRENTS

Folding, assembly and trafficking of ion channels are tightly controlled processes and important for biological functions relevant to health and disease. Here, we report that functional expression of the Eag channel is temperature-sensitive by a mechanism that is independent of trafficking or surface targeting of the channel protein. Eag channels in cells grown at 37 0 C exhibit voltage-evoked gating charge movements, but fail to conduct K + ions. By mutagenesis and chimeric channel studies, we show that the N- and C-terminal regions are involved in controlling a step after movement of the voltage sensor, as well as in regulating biophysical properties of the Eag channel. Synthesis and assembly of Eag at high temperature disrupts the ability of these domains to carry out their function. These results suggest an important role of the intracellular regions in generation of Eag currents.

Folding, assembly and trafficking of ion channels are tightly controlled processes and important for biological functions relevant to health and disease. Here, we report that functional expression of the Eag channel is temperature-sensitive by a mechanism that is independent of trafficking or surface targeting of the channel protein. Eag channels in cells grown at 37 0 C exhibit voltage-evoked gating charge movements, but fail to conduct K + ions. By mutagenesis and chimeric channel studies, we show that the N-and C-terminal regions are involved in controlling a step after movement of the voltage sensor, as well as in regulating biophysical properties of the Eag channel. Synthesis and assembly of Eag at high temperature disrupts the ability of these domains to carry out their function. These results suggest an important role of the intracellular regions in generation of Eag currents.
Voltage-gated K + channels consist of a large family of proteins that selectively conduct potassium ions when activated by changes of membrane potential (1). They are comprised of four subunits, each containing six transmembrane segments (S1-S6) that assemble together to form the voltage sensor and central pore domains (2,3). It is now well established that opening of these channels is initiated by conformational changes of the voltage sensor domains upon membrane depolarization. Little, however, is known regarding the molecular and physical mechanisms underlying the coupling between the voltage sensor and activation gate of these K + channels (4). Voltage-gated K + channels diverge significantly in their N-and C-terminal (NT and CT) regions, and for many channels these domains have been shown to be important for binding of auxiliary subunits or modulators (5). The roles of intracellular domains in gating, activation and inactivation are less well understood (6)(7)(8).
The Drosophila ether-à-go-go channel (Eag) is the founding member of an evolutionarily conserved subfamily of voltage-gated K + channels, which includes HERG (human eag-related gene), a channel that plays an important role in regulating cardiac excitability and maintaining normal cardiac rhythm (9,10). HERG channel dysfunction is responsible for a third of congenital long QT syndrome and the vast majority of drug-induced long QT (10). In flies, mutation of eag increases neuronal excitability and affects memory formation (11,12). This family of channels is therefore critical to the function of multiple types of excitable cells across the animal kingdom.
Structurally, Eag family channels are related to both voltage-gated K + channels and cyclic nucleotide-gated cation channels as they not only have transmembrane domains for voltage sensing and K + ion permeation but also contain a cyclic nucleotide binding motif within their carboxyl terminal regions (13,14). While Eag family channels may not be directly gated by cyclic nucleotides, several lines of evidence have suggested their NT and CT domains are important to channel function. First, mutations in these regions can result in defective synthesis, trafficking, or gating of the channels. Such mutations in HERG channels are one of the most prevalent causes of congenital human long QT syndrome (15,16). Second, Eag channel activity and/or surface expression are regulated by a number of signaling molecules through direct binding to the NT and CT domains (17)(18)(19)(20). Moreover, the eag transcript can be alternatively spliced to produce a protein that contains only the NT and CT regions and modulates cellular signal transduction pathways (21).
In this study, we report a direct involvement of these intracellular regions in the activation of Eag K + channels. Our data reveal that, in heterologous expression systems, the NT and CT regions of Eag are critical for functional expression of K + currents, specifically processes following movement of the voltage-sensing domain.

EXPERIMENTAL PROCEDURES
Plasmids and construction -The cDNA encoding Drosophila Eag (kindly provided by Dr. Gisela Wilson, UW Madison) was subcloned into a mammalian expression vector, pcDNA3. The myc-eag and eag-EGFP cDNAs were constructed by either cloning the eag coding sequence into the pCS2 vector containing six myc epitopes, or fusing EGFP to the carboxyl terminus of the eag coding region in the pEGFP-N1 vector. To measure surface expression of Eag proteins, a myc tag was inserted in the extracellular loop that connects transmembrane domains S1 and S2 of the Eag channel (Eag-EX-myc). The insertion changes the amino acid sequence between S1 and S2 to 255 PYNVAFKEQKLISEEDLNKTSEDVS, in which the myc epitope is shown in bold type. The chimeric Eag-mEag channels (Eag-NTmEag, Eag-CTmEag or Eag-TMmEag) were created by replacing the N-, C-terminal tail or transmembrane region of Drosophila Eag (residues 1-211, 501-1174 or 212-500) with corresponding region of the mouse Eag channel (residues 1-192, 509-987 or 193-508). A similar approach was utilized to generate N-terminal truncation mutants of Eag or mEag. The mEag NT domain (mEagNT1-190-HA) was cloned into the pIRES-EGFP vector. The C-terminal truncation mutants of wild-type Eag, mEag or chimeric Eag were constructed by introducing a stop codon into the cDNA at appropriate locations using the Quickchange strategy (Stratagene). All cDNA constructs were verified by DNA sequencing in the FSU Biology sequencing facility, Tallahassee, FL. Biotinylation of cell-surface proteins -eag-pcDNA3 was transiently expressed in tsA201 cells using a calcium phosphate-mediated transfection protocol. 2 days after transfection, cells were incubated with 0.5 mg/ml EZ-link-Sulfo-NHS-LCbiotin (Pierce Chemical Co.) in PBS, pH 8.0, at room temperature for 30 min with gentle agitation. After removing the reagent by washing cells three times with PBS, cells were harvested and lysed using standard protocols (22). Biotinylated proteins were precipitated from cell lysates with ImmunoPure streptavidin beads (Pierce). Channel proteins in total cell lysates and streptavidin precipitates were analyzed by immunoblot using an affinity purified anti-Eag antibody as described previously (20). To confirm that the biotinylation reagent did not leak into the cell and label intracellular proteins, we stripped and reprobed the same blots with an anti-GAPDH antibody (EnCor Biotechnology, Inc.). Immunoblot images were quantified using ImageJ 1.43 (http://rsb.info.nih.gov/nih-image/). Confocal microscopy -tsA 201 or Chinese hamster ovary (CHO) cells were grown on poly-Llysine-treated cover glasses and transfected with eag-EX-myc cDNAs. To label cell surface Eag proteins, cells were first incubated with 20 ug/ml anti-myc antibody (Millipore) for 30 min at 37 0 C, and then fixed with 4% paraformaldehyde in PBS (30 min), blocked in 5% goat serum in PBS with 0.1% Triton X-100 (30 min), and labeled with FITC-conjugated anti-mouse antibody (2 h). To visualize total Eag proteins, transfected cells were first fixed and permeabilized with 0.2% Triton, and the incubated with anti-Eag antibody and FITC-conjugated anti-rabbit antibody in the presence of 10% goat serum. Fluorescence images were acquired with a Leica TCS SP2 AOBS laser confocal microscope and analyzed using the ImageJ 1.43 software.
Electrophysiology -Ionic and gating currents of Eag channels were measured in the whole-cell configuration from CHO or tsA 201 cells transiently transfected with various eag cDNAs along with the pEGFP-N1 vector cDNA (Clontech) in a 9:1 ratio using either the Fugene 6 (for tsA 201 cells; Roche, Indianapolis, IN) or Lipofectamine LTX (for CHO cells; Invitrogen) transfection reagents. Transfected cells bearing GFP fluorescence were identified with a FITC filter set on a TE2000U inverted fluorescence microscope (Nikon). Patch electrodes with resistances of 2-4 MΩ were pulled from borosilicate glass and fire-polished. Ionic and gating currents were digitized at 20 kHz and filtered at 1 and 5 kHz respectively with an Axopatch 200B amplifier. Data acquisition and analysis were performed with pCLAMP10 software (Molecular Devices). To record gating currents, a P/4 protocol was applied on-line to subtract leak and capacitive currents (23).
For ionic K + current recording, the standard bath solution contained 2 mM KCl, 148 mM NaCl, 2 mM MgCl 2 , 10 mM HEPES and 1 mM EGTA, pH 7.4. The pipette solution contained 130 mM KCl, 10 mM HEPES and 5 mM EGTA, pH 7.4. For gating currents measurements, the extracellular Na + and intracellular K + ions were replaced with equal molar of either NMG + or TEA-Cl. Recordings were generally conducted at room temperature. In experiments involving acute temperature changes, the bath temperature was maintained by a heated stage with the temperature controller, TC-344B (Warner Instruments).
The channel conductance was determined by measuring the amplitudes of isochronal tail currents evoked by repolarizing to -60 mV from various depolarizing potentials. Tail current amplitudes were normalized to the maximum value obtained in the experiment and plotted verse depolarizing potentials. Data were fitted with a single Boltzmann function using Origin 7.0 (Origin Lab, Northampton, MA). Deactivation kinetics was measured by fitting the tail currents with a single exponential function to obtain values for the deactivation time constant (τ deact ) using the Clampfit 10 program (Molecular Devices). As Eag channel currents display sigmoid rise phase, activation kinetics was quantified by measuring the time to half maximal current amplitude (t 1/2 ) at a depolarizing potential of +60 mV.
Temperature change protocol -tsA 201 or CHO cells were maintained at 37 0 C. For experiments performed at 26 0 C, cells were kept at 37 0 C for 24 hrs following transfection, and then switched to an incubator maintained at 26 0 C. Data analysis -The majority of data were analyzed statistically by one-way ANOVA using Origin 7 software. Statistical significance was set at P < 0.05. To compare the probability of gating currents occurrence between groups, we used standard logistic regression analysis using the statistical software R-2.11.1.

RESULTS
Functional expression of recombinant Eag K + channels is dependent upon cultivation temperature. Eag produces voltage-dependent outward K + currents when expressed in Xenopus oocytes (24). In contrast, when eag cDNA is transfected into cultured mammalian cells, no significant macroscopic K + current is elicited by depolarizing voltage steps from either transfected CHO or tsA 201 cells ( Fig. 1A & B, middle panels). Mammalian cell lines differ from Xenopus oocytes in their culture temperature (37 0 C), which is known to affect trafficking and/or surface targeting of some ion channels including mutant HERG channels (25)(26)(27)(28). To determine if temperature was affecting current levels, we measured whole-cell K + currents from mammalian cells that were cultivated at lower temperatures after transfection with eag cDNA. As shown in Figures 1A & B (right panels), cultivation at 26 0 C for 24 h resulted in production of voltagedependent macroscopic K + currents in both CHO and tsA 201 cells transfected with eag cDNA. Similar to Eag K + currents recorded from Xenopus oocytes, the whole-cell K + currents seen after growth at 26 0 C were activated around -40 mV and exhibited open-state inactivation at depolarized voltages. In addition, activation kinetics of the K + currents were slowed by either increasing the concentration of extracellular Mg 2+ or applying a hyperpolarizing prepulse potential (29), both of which are characteristic features of Eag channels (28,30).
To assess the effect of cultivation temperature on current expression of Eag channels, we measured whole-cell K + currents in eag-transfected CHO cells after cultivating at various temperatures for 24 h. As quantified by current density (pA/pF) at a depolarizing potential of +40 mV, macroscopic K + currents began to be restored by lowering cultivation temperature to 30 0 C, while the largest augmentation of current level was observed at 26 0 C (Fig. 1C). At 26 0 C, restoration of macroscopic K + currents required cultivation of transfected cells for a minimum of 3 h, and the level of Eag K + currents was substantially increased by longer cultivation at the lower temperature (Fig. 1C). Consistent with these results, recording at room temperature for extended period did not restore currents from cells cultivated at 37 0 C, nor did acute up-shift of temperature to 37 0 C during recordings affect the rate of rundown or abolish K + currents in cells grown at 26 0 C (Fig. 1D). Together, these data suggest that temperature-dependent expression of Eag currents is not a direct effect of recording temperature on channel activation. Surface expression of Eag channels is not affected by cultivation temperatures. One explanation for the restoration of macroscopic K + currents by lower temperature could involve temperaturesensitivity of the cell surface expression of the channel. To test this, we assessed the effect of culture temperature on plasma membrane targeting of Eag proteins using surface biotinylation with avidin pull down to selectively harvest Eag proteins that were on the plasma membrane. Similar to previous reports, the anti-Eag antibody identifies two bands from both avidin pull down and total cell lysates ( Fig. 2A) which correspond to the full length channel and an 80 kDa protein (Eag80) which was produced from a cryptic inframe splicing event and occurs with transfections of the full length eag cDNA (21). Surface expression of Eag channels was not affected by cultivation temperature, as the ratio of biotinylated Eag proteins to those in total lysates was not significantly different between cells cultivated at 26 0 C and 37 0 C (Fig. 2B). As a control, we also examined surface targeting of a chimeric Eag-NTmEag channel whose current expression was independent of cultivation temperature (see Fig.  5). For both Eag and Eag-NTmEag, the full length proteins are identically sized tight bands at 26 0 C and 37 0 C ( Fig. 2A), suggesting that there was no temperature-dependent shift in the molecular weight of channel proteins in these cells that could reflect differential glycosylation or other maturation processes.
We next directly visualized the surface expression of Eag proteins using immunofluorescence. In these experiments, surfacetargeted Eag channels were probed with an antimyc antibody that recognized a myc epitope inserted on the extracellular side of the Eag channel, and visualized with FITC-conjugated secondary antibody by confocal microscopy. In Figure 2C (top panels), it can be seen that Eag channels were localized to the plasma membrane in transfected cells cultured at both 37 0 C and 26 0 C. When the cell membranes were permeablized with non-ionic detergent, we did not observe any significant difference in the cytoplasmic distribution of total Eag proteins between cells cultivated at 26 0 C and 37 0 C (Fig. 2C, bottom panels). Together, these data demonstrate that the temperature-dependent expression of K + channel currents can not be attributed to a deficit in trafficking or surface targeting of Eag channels. Voltage-dependent gating charge movement of Eag K + channels is not affected by cultivation temperatures. Voltage-dependent opening of ion channels is a process that entails coordinated conformational changes of both the voltage sensor and the activation gate (4). To identify the defective step(s) that might have caused the failure to conduct K + ions when the Eag channel is assembled and targeted at 37 0 C, we assessed the integrity of its voltage sensor by measuring voltage-dependent gating charge movements. To record Eag gating currents, we used intracellular and extracellular NMG + solutions to substitute for K + and Na + ions and applied a P/-4 voltage protocol to subtract capacitive and leak currents. As shown in Figure 3A, on-and off-gating currents were elicited by depolarizing voltages in some eag-transfected CHO cells cultivated at either 26 0 C or 37 0 C. These were genuine gating currents of Eag channels, since they were not present in any untransfected or vector-transfected CHO cells (Fig. 3B) (31), and were similar in voltage dependency and kinetics to that of the Eag on-and off-gating currents previously recorded in oocytes (30,32). However, unlike Shaker channels that exhibited ample gating currents in a majority of transfected cells, Eag gating currents had smaller amplitudes and well-resolved on-and offgating currents were only detected above noise in a fraction of eag-transfected cells (Fig. 3B). Importantly, the percentage of cells that exhibited measurable gating currents was not different in transfected cells cultivated at the two temperatures (Fig. 3B), and a comparison of the charge-voltage relationship (Q-V) also revealed little difference between Eag gating currents recorded from cells cultivated at 26 0 C and 37 0 C (Fig. 3C).
We also recorded Eag gating currents using intracellular and extracellular solutions containing a K + channel blocker, TEA. In eag-transfected cells, this recording condition yielded sizeable ongating but very small off-gating currents, which is consistent with the short pulse duration and the presence of TEA in these experiments (23,30). Different from on-gating currents of Shaker K + channels, Eag on-gating currents were activated at higher depolarizing voltages and had a slower rising phase (Fig. 3D). The kinetics and voltagedependency of the on-gating currents were not significantly different for eag-transfected cells cultivated at 26 0 C and 37 0 C, confirming that cultivation temperatures did not affect voltagedependent gating charge movements of the Eag channel. Intracellular terminal regions play a significant role in structure and function of Eag K + channels. The Eag protein shares substantial homology with its mammalian counterparts (Fig. 4), but functional expression of other Eag homologues is not dependent on cultivation temperatures (14). To determine the potential role of various regions of the Eag channel in mediating temperaturedependent expression of functional K + channels, we employed a chimeric channel strategy. We first generated three chimeric channels (Eag-S1-S6mEag, Eag-CTmEag and Eag-NTmEag) in which either the transmembrane (S1-S6), carboxyl-(CT) or amino-terminal (NT) domain of the Eag channel was replaced with the corresponding region of its mouse homologue, mEag (Fig. 4). mEag transfected cells have robust K + currents regardless of culture temperature (Fig.  5A). When either its NT or CT was replaced with mEag sequences, the temperature-dependency of Eag current was alleviated. Robust K + currents were elicited from transfected cells at both temperatures ( Fig. 5B & 5C), although the current density was higher in cells maintained at 26 0 C than that at 37 0 C (Fig. 5E). In addition, the chimeric channels exhibited changes in their biophysical properties. The Eag-NTmEag channel had slower activation kinetics than wild-type Eag (Fig. S1A), whereas the Eag-CTmEag channel had an enhanced voltage sensitivity (Fig. 5F) and considerably slower deactivation kinetics than Eag and Eag-NTmEag channels (Fig. 5G).
One possible interpretation of the finding that replacement of either the NT or CT of Eag with mEag was sufficient to restore currents at 37 0 C is that Eag channel assembly at high temperature disrupts some interaction between these two domains that blocks their ability to participate in allowing opening of the channel following movement of the voltage sensor. Substitution of the mEag NT or CT allows a functional interaction between terminal domains and rescues activation. To further probe the role of the interaction of the transmembrane domain with the intracellular domains in activation, we generated a chimeric channel that contained both the Eag NT and CT attached to the mEag transmembrane domains. Although it was expressed at the cell surface (Fig.  S2), the Eag-S1-S6mEag channel failed to produce any significant K + currents when it was transfected into mammalian cells cultivated at either 26 0 C or 37 0 C (Figs. 5D & 5E). Hence, while this experiment confirmed the importance of intracellular domains for function, the failure of Eag NT and CT domains to support activation of the mEag channel suggested that there were significant differences in how the TM regions interacted with their cognate intracellular domains.
Comparison of the fly and mouse Eag channels sequences reveals that only the proximal regions of their CT domains (amino acid residues 496-734 of Eag and 504-743 of mEag) show good conservation (Fig. 4). To test the differential roles of the distal and proximal CT domains, we generated Eag and mEag channels which have a truncation of the distal CT sequence where Eag and mEag are least homologous (Fig. 4). Notably none of the channels (mouse, fly and chimera) that lack the distal CT domain produced current at 37 o C and all had greatly reduced current amplitudes at 26 o C ( Fig. 6; note difference in Y axis scale between Fig. 6G and Fig. 5E). This indicates a general role for the distal CT in modulation of current amplitude.
The distal CT also appeared to have additional functionality. When transfected into mammalian cells, the Eag∆735-1174 channel was not much different from the full length wild-type channel in its temperature-dependent conduction of K + ions (Fig. 6A). In contrast, this C-terminal deletion converted the non-conducting chimeric channels that had the mEag transmembrane domains into an Eag-like K + channel. Eag-S1-S6mEag∆735-1174 channels produced macroscopic K + currents, but only when the transfected cells were cultivated at lower temperatures (Fig. 6B), implying that the distal CT was responsible for the inability of the Eag-TMmEag channel (Fig. 5D) to conduct K + .
Interestingly, after a deletion of the equivalent distal C-terminal region, the two temperatureindependent channels, mEag and Eag-CTmEag, became temperature sensitive, as both mEag∆744-987 and Eag-CTmEag∆744-987 only generated K + currents when cultivated at lower temperatures (Figs. 6C & 6D), suggesting another role for the distal CT in stabilization of the NT/proximal CT interaction. Removal of the entire CT domain (Eag ∆502-1174) rendered the fly Eag channel nonconductive at either temperature (Fig. 6E). In contrast, the mutant mEag channel devoid of Cterminal tail (mEag∆510-987) was capable of producing a low level of macroscopic K + currents at 26 o C (Fig. 6F). The proximal CT domain appears to be absolutely required in the fly, but not in the mouse for ion conductance. In addition, these CT-truncated mutant channels had slower activation kinetics when compared to the respective full length channels (Fig. S1B), indicating an involvement of CT domains in channel biophysical property.
We also examined the role of the NT domain in current expression by constructing mutant Eag and mEag channels which have truncations of their N-terminal regions. Previously, it had been reported that, when expressed in Xenopus oocytes, NT-truncated rat Eag and HERG channels produce functional K + currents with altered activation or inactivation properties (33), which can be restored by co-expression of a recombinant N-terminal domain (34,35). When transfected into CHO cells cultivated at 37 0 C, the NT-truncated mutant mEag channel (mEag∆2-188) yielded very small currents; only 6 in 14 transfected cells exhibited detectable macroscopic currents with an average current density of 5.77 pA/pF (Fig. 7B). Current levels in cells transfected with mEag∆2-188 were significantly enhanced by cultivating cells at lower temperature; the average current density increased to 35.01 pA/pF in cells cultivated at 26 0 C. We next tested the effect of a recombinant mEag NT domain on the NT-truncated mEag channel. As shown in Fig. 7D, co-expression of mEag∆2-188 and mEagNT1-190 in cells cultivated at 26 0 C led to generation of robust K + currents which were similar to wild-type mEag in both current density and deactivation kinetics. Intriguingly, the enhancement of current expression by mEag NT domain was not observed in cells cultivated at 37 0 C (Fig. 7D, left trace).
The NT domain of Eag is largely homologous with that of mEag (Fig. 4). In this study, we generated three mutant Eag channels which are truncated at amino acids 30, 97 and 122 of its NT domain. Using biochemical approaches, we verified that these mutant channel proteins were expressed and targeted to cell surface (Fig. S2). However, they failed to produce any K + currents in transfected CHO cells (Fig. 7A), nor did coexpression of the recombinant mEag NT domain restore current expression at different cultivation temperatures (Fig. 7C). Thus, integrity of the Eag NT domain appears to be critical for its functional expression.
To obtain additional evidence in support of the role of the Eag NT and CT domain interaction in channel activation processes, we examined the effect of N-or C-terminal tagging on Eag channel conductivity. Addition of such protein tags can disrupt native interactions in proteins (36,37). As shown in Figs. 6G & 7E, no voltage-activated K + current was recorded in cells that were transfected with either a tandem of six N-terminal myc (myc-Eag) or a C-terminal EGFP (Eag-EGFP) tagged channel. The failure of conduction was not caused by a deficit in surface expression (Fig. S2), and could not be rescued by lowering the cultivation temperature to 26 0 C. This is consistent with a previous report showing that a similar myc-tagged Eag channel construct produces detectable gating currents but not outward K + currents when expressed in Xenopus oocytes (38). These results indicate that addition of amino acids to either the NT or CT of the Drosophila Eag channel is sufficient to disrupt their functional interaction.

DISCUSSION
The Drosophila Eag channel was originally identified based on the phenotype of a mutant with ether-induced leg-shaking (39). This led to the discovery of a family of related K + channels with a variety of biological functions (13). However, unlike most other families of K + channels cloned from Drosophila, Eag channels fail to produce voltage-dependent K + currents in cultured mammalian cells, a widely used heterologous expression system that is advantageous for both biochemical and electrophysiological studies of ion channels. In this report, we demonstrate that the Eag channel can be functionally expressed in mammalian cell lines at lower cultivation temperatures. We show that voltage-dependent gating charge movements are preserved for Eag channels assembled and targeted at 37 0 C. Therefore, absence of K + currents in these channels has to be caused by a defect in one of the subsequent processes, such as coupling between voltage sensor and activation gate and opening of the ion conduction pore (4,8).
The mechanism by which lower cultivation temperature restores Eag channel functions appears to be a novel one. It is known that reducing culture temperature can facilitate surface expression of trafficking-defective membrane proteins, including mutant CFTR and HERG channels (26,40). Using both biochemical and immunocytochemical approaches, we have determined that surface expression of Eag proteins is not significantly affected by cultivation temperatures. Temperature may affect many other steps of channel protein biogenesis including hydrophobic interactions in protein folding (16,41,42). Once Eag channels are inserted into the plasma membrane, however, their conformations are minimally affected by temperature, since Eag K + currents persist when recorded at 37 0 C, and restoring K + currents requires cultivation of transfected cells at a lower temperature for several hours, which presumably reflects time required for new channel biogenesis and assembly. The fact that gating charge movements of Eag channels were independent of cultivation temperature suggests that the positioning of the TM domains that comprise the voltage sensor is intact. This raises the possibility that the temperature-dependence of Eag channel properties is reporting a difference in the conformation of Eag intracellular domains or their interface with the TM domains. Hence, our results provide an apparent example of subtle temperature-dependent effects on potassium channel biosynthesis and assembly, in contrast to the more usual catastrophic effects of misfolding which result in complete retention of nascent channel proteins in ER or Golgi.
The ability of the structure of the NT and CT domains to confer both temperature sensitivity and to activate ion conductance indicate that these domains have a critical role in regulation of Eag activity. Our results suggest a model in which the N-and proximal C-termini of Eag interact with each other to regulate channel activation. This regulatory function is disrupted by folding at 37 0 C, a temperature not normally experienced by this protein since D. melanogaster is not viable for long in this temperature range. Interestingly, removal of particular domains of mEag can also generate a temperature-sensitive channel, suggesting that these NT/CT interactions are a conserved feature of this family. Our assessment of multiple deletion and chimeric channels indicates that the temperature sensitivity of Eag does not map to a specific residue or domain, but rather appears to be due to a failure at the level of domain interaction. These analyses also point to specific functions for the NT, proximal CT and distal CT domains in this collaboration.
Sequences in the unconserved distal CT functionally distinguish between fly and mouse TM domains, perhaps implying that residues within the TM domains or in the short intracellular loops are critical for brokering the effects of the intracellular domains on activation. The distal CT is also clearly involved in modulation of current amplitude, since all channels that lack this subdomain have substantially reduced amplitudes. The ability of deletion of the distal CT to make mEag temperature sensitive might suggest a third role as a stabilizer for NT/proximal CT/channel interactions at higher temperatures. The proximal CT, on the other hand, appears to have disparate roles in the mouse and the fly. In the fly channel it is absolutely required, while mEag channels that lack all CT sequences can still pass small currents. This perhaps is consistent with a role for this subdomain to help position the NT domain for channel activation.
The role of the NT domain also differs between Eag and mEag. It is absolutely required in the fly channel, since even small deletions abolish currents. mEag channels lacking this domain are still able to pass current, but do so with greatly decreased amplitudes, especially at 37 o C. Providing the mEag NT in trans fully rescues NTless mEag at 26 o C, but not at 37 o C. Coexpression of mEag NT does not reinstate the ability of NTless Eag to conduct ions. This differential rescue supports a model that full coupling of gating to activation requires a multi-domain complex of the NT and CT and would be consistent with the idea that the intrinsic binding of these domains in Eag is weaker than in mEag. When the NT is attached to the Eag or mEag channel domain, it is in very high local concentration and this drives binding between NT and CT. When the NT is provided as a separate protein, its interaction with the CT is now diffusion-limited. In the case of the mouse channel at low temperatures, the complex is stable and you see current at 26 o C. Higher temperatures destabilize the complex. In the case of the fly channel, the NT/CT binding is very weak and unstable in the diffusion-limited case even at 26 o C. This would make the prediction that driving binding by providing very high levels of mEag NT in trans or on might rescue the NT-less Eag. The hypothesized differential temperature sensitivities of the fly and mouse NT/CT interactions could be a reflection of the different temperatures normally experienced by temperate-living poikilotherms and warm-blood homeotherms.
The coupling of gating to activation is not the only role for these intracellular domains. Our studies using chimeric Eag-mEag channels also revealed significant contributions of the NT and CT regions to determining both the voltagedependence of activation and activation and deactivation kinetics, confirming that these intracellular regions are integral to multiple aspects of Eag channel function (43).
Intrigued by the temperature-dependent functional expression of Eag channels, we have identified an important role of the intracellular domains in Eag channel structure and function. Future studies using more sophisticated methodologies should be able to pin down the temperature-sensitive interactions, and thus gain insight into the molecular details of Eag channel activation. As the NT and CT regions are sites of interactions for several regulatory proteins, our findings may shed some light on the functional significance of these modulatory protein complexes. In particular, they may bear some relevance to the signaling roles of Eag family channels that are suggested to correlate with channel conformation and the state of activation (38,44,45). On the other hand, our dada may help to elucidate certain types of channelopathies resulted from production of non-conducting channels which are trafficked to the plasma membrane. This is particularly applicable to human long QT syndrome, which can be caused by defective activation of Herg channels due to mutations in the intracellular domains of the channel protein. Moreover, our findings may provide a potential model for non-conducting Eaglike channels induced by environmental stress, such as higher temperatures experience by patients at pathological conditions.  Recording at 37 0 C does not abolish Eag K + currents. Cells were repeatedly depolarized to +40 mV from a holding potential of -80 mV. Solid bar denotes the period during which recording temperature was raised to 37 0 C, and data at this condition are represented by filled squares (mean ± SEM, n=3). There was a comparable rundown of Eag currents when recordings were conducted throughout at room temperature (RT, [22][23][24][25]      Comparison of the deactivation kinetics of Eag and the two chimeric channels, measured as the deactivation time constant (τ deact ) (mean ± SEM). Unlike Eag-NTmEag, the Eag-CTmEag channel deactivates significantly slower than Eag (**P < 0.01, one-way ANOVA).  Quantification of current density (mean ± SEM) at +40 mV from CHO cells transfected with various NT truncated or N-terminal myc tagged channels cultivated at either 37 0 C or 26 0 C (*P < 0.05, **P < 0.01, one-way ANOVA). Data of wild type mEag channels were included for comparison. Dotted line indicates current density of zero. Note that no current was expressed when Eag protein was fused with six Nterminal myc tags.