The predicted TM10 transmembrane sequence of the cardiac Ca2+ release channel (ryanodine receptor) is crucial for channel activation and gating

The predicted TM10 transmembrane sequence, (4844)IIFDITFFFFVIVILLAIIQGLII(4867), has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca(2+). Intracellular Ca(2+) release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca(2+) dependence of [(3)H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca(2+) and enhanced the basal activity of [(3)H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca(2+). Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore.     

The pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels

Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K(+) ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.     

Genetic screen for potassium leaky small mechanosensitive channels (MscS) in Escherichia coli: recognition of cytoplasmic β domain as a new gating element

Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore.     

Role of conserved glycines in pH gating of Kir1.1 (ROMK)

Gating of inward rectifier Kir1.1 potassium channels by internal pH is believed to occur when large hydrophobic leucines, on each of the four subunits, obstruct the permeation path at the cytoplasmic end of the inner transmembrane helices (TM2). In this study, we examined whether closure of the channel at this point involves bending of the inner helix at one or both of two highly conserved glycine residues (corresponding to G134 and G143 in KirBac1.1) that have been proposed as putative "gating hinges" for potassium channels. Replacement of these conserved inner helical glycines by less flexible alanines did not abolish gating but shifted the apparent pKa from 6.6 +/- 0.01 (wild-type) to 7.1 +/- 0.01 for G157A-Kir1.1b, and to 7.3 +/- 0.01 for G148A-Kir1.1b. When both glycines were mutated the effect was additive, shifting the pKa by 1.2 pH units to 7.8 +/- 0.04 for the double mutant: G157A+G148A. At this pKa, the double mutant would remain completely closed under physiological conditions. In contrast, when the glycine at G148 was replaced by a proline, the pKa was shifted in the opposite direction from 6.6 +/- 0.01 (wild-type) to 5.7 +/- 0.01 for G148P. Although conserved glycines at G148 and G157 made it significantly easier to open the channel, they were not an absolute requirement for pH gating in Kir1.1. In addition, none of the glycine mutants produced more than small changes in either the cell-attached or excised single-channel kinetics which, in this channel, argues against changes in the selectivity filter. The putative pH sensor at K61-Kir1.1b, (equivalent to K80-Kir1.1a) was also examined. Mutation of this lysine to an untitratable methionine did not abolish pH gating, but shifted the pKa into an acid range from 6.6 +/- 0.01 to 5.4 +/- 0.04, similar to pH gating in Kir2.1. Hence K61-Kir1.1b cannot function as the exclusive pH sensor for the channel, although it may act as one of multiple pH sensors, or as a link between a cytoplasmic sensor and the channel gate. K61-Kir1.1b also interacted differently with the two glycine mutations. Gating of the double mutant: K61M+G148A was indistinguishable from K61M alone, whereas gating of K61M+G157A was midway between the alkaline pKa of G157A and the acid pKa of K61M. Finally, closure of ROMK, G148A, G157A, and K61M all required the same L160-Kir1.1b residue at the cytoplasmic end of the inner transmembrane helix. Hence in wild-type and mutant channels, closure occurs by steric occlusion of the permeation path by four leucine side chains (L160-Kir1.1b) at the helix bundle crossing. This is facilitated by the conserved glycines on TM2, but pH gating in Kir1.1 does not absolutely require glycine hinges in this region.     

The (beta)gamma subunits of G proteins gate a K(+) channel by pivoted bending of a transmembrane segment

The molecular mechanism of ion channel gating remains unclear. Using approaches such as proline scanning mutagenesis and homology modeling, we localize the gate of the K(+) channels controlled by the (beta)gamma subunits of G proteins at the pore-lining bundle crossing of the second transmembrane (TM2) helices. We show that the flexibility afforded by a highly conserved glycine residue in the middle of TM2 is crucial for channel gating. In contrast, flexibility introduced immediately below the gate disrupts gating. We propose that the force produced by channel-G(beta)gamma interactions is transduced through the rigid region below the helix bundle crossing to bend TM2 at the glycine that serves as a hinge and open the gate.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels

P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration-response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting alpha-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Redox-dependent gating of G protein-coupled inwardly rectifying K+ channels

G protein-coupled inwardly rectifying K(+) channels (GIRK) play a major role in inhibitory signaling in excitable and endocrine tissues. The gating mechanism of these channels is mediated by a direct interaction of the Gbetagamma subunits of G protein, which are released upon inhibitory neurotransmitter receptor activation. This gating mechanism is further manifested by intracellular factors such as anionic phospholipids and Na(+) and Mg(2+) ions. In addition to the essential role of these components for channel function, phosphorylation events can also modulate channel activity. In this study we explored the involvement of redox modulation on GIRK channel function. Extracellular application of the reducing agent dithiothreitol (DTT), but not reduced glutathione, activated GIRK channels without affecting their permeation or rectification properties. The DTT-dependent activation was found to mimic receptor activation and to act directly on the channel in a membrane delimited fashion. A critical cysteine residue located in the N-terminal cytoplasmic domain was found to be essential for DTT-dependent activation in hetero- and homotetrameric contexts. Interestingly, when mutating this cysteine residue, DTT-dependent activation was abolished, but receptor-mediated channel activation was not affected. These results suggest that intracellular redox potential can play a major role in tuning GIRK channel activity in a receptor-independent manner. This sort of redox modulation can be part of an important cellular protective mechanism against ischemic or hypoxic insults.     

Yeast screen for constitutively active mutant G protein-activated potassium channels

GIRK2 is a major contributor to G protein-activated inward rectifier potassium channels in the mammalian brain. How GIRK channels open upon contact with Gbetagamma remains unknown. Using a yeast genetic screen to select constitutively active mutants from a randomly mutagenized GIRK2 library, we identified five gating mutations at four residues in the transmembrane domain. Further mutagenesis indicates that GIRK channel opening involves a rotation of the transmembrane segments, bringing one of these residues (V188) to a pore-lining position in the open conformation. Combined with double-mutant studies, these findings suggest that GIRK channels gate by moving from the open conformation inferred from our yeast study of Kir2.1 to a closed conformation perhaps resembling the known KcsA structure.     

Gating of voltage-dependent potassium channels

Activation of voltage-dependent ion channels is primarily controlled by the applied potential difference across the membrane. For potassium channels the Drosophila Shaker channel has served as an archetype of all other potassium channels in studies of activation mechanisms. In the Shaker potassium channel much of the voltage sensitivity is conferred by the S4 transmembrane helix, which contains seven positively charged residues. During gating, the movement of these charges produces gating currents. Mutagenic and fluorescence studies indicate that four of these residues are particularly important and contribute to the majority of gating charge, R362, R365, R368 and R371. The channel is thought to dwell in several closed states prior to opening. Ionic-charge pairing with negatively charged residues in the S2 and S3 helices is thought to be important in regulating these closed states and detailed kinetic models have attempted to define the kinetics and charge of the transitions between these states. Neutral residues throughout the S4 and S5 helices are thought to control late steps in channel opening and may have important roles in modulating the stability of the open state and late closed states. In response to depolarization, the S4 helix is thought to undergo a rotational translation and this movement is also important in studies of the movement of the pore helices, S5 and S6, during opening. This review will examine residues that are important during activation as well as kinetic models that have attempted to quantitatively define the activation pathway of voltage-dependent potassium channels.     

Proline-induced hinges in transmembrane helices: possible roles in ion channel gating

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.     

Capping transmembrane helices of MscL with aromatic residues changes channel response to membrane stretch

Tyrosines and tryptophans that anchor both ends of the helices to membrane interfaces in many transmembrane proteins are not common in MscL and homologous mechanosensitive channels. This characteristic absence of two aromatic "belts" may be critical for MscL function as the opening transition is predicted to be associated with a strong helical reorientation. A single tyrosine (Y75) on the extracellular side of the M2 helix of pentameric EcoMscL is absent in TbMscL, which instead has a single tyrosine (Y87) on the cytoplasmic side of M2. Moving the tyrosine of EcoMscL to the intracellular side (Y75F/F93Y) or capping the TM2 helix on both sides (F93Y/W) slows the kinetics of gating and increases the threshold for activation, leading to a partial loss-of-function in osmotic shock survival assays. Increasing the distance between the caps (L98W, L102Y/W) partially restores channel function presumably by loosening restraints for tilting. Capping the TM2 helix with a charged residue (Y75E) causes a right shift of the activation curve ("stiff" phenotype) and abolishes function. Introducing a "cap" into the TM1 helix (I41W) decreases the activation threshold and shortens the mean open time but unexpectedly leads to a complete loss-of-function in vivo. The data are consistent with the view that restraining helical positions in MscL by introducing specific protein-lipid interactions at membrane interfaces compromises MscL function. Subtle differences in osmotic shock survival are more evident at low levels of mutant protein expression. We observed a correlation between the right shift of tension activation threshold and the loss-of-function channel phenotype, with a few exceptions that point to other parameters of gating that may define the osmotic rescuing ability in vivo.     

Coupling Gbetagamma-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels

G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gbetagamma subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gbetagamma. Mutations in TM2 were found that mimicked the Gbetagamma-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gbetagamma subunits but depended on PtdIns(4,5)P(2). The results suggest that the TM2 region plays a key role in channel gating following Gbetagamma binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.     

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.     

C-Linker of Cyclic Nucleotide–gated Channels Controls Coupling of Ligand Binding to Channel Gating

Cyclic nucleotide–gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K⁺ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by ∼90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide–gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.     

Structural basis for modulation of gating property of G protein-gated inwardly rectifying potassium ion channel (GIRK) by i/o-family G protein α subunit (Gαi/o)

G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein βγ subunits (Gβγ), which are released from pertussis toxin-sensitive G protein α subunit (Gα(i/o)) upon GPCR activation in vivo. Several lines of evidence indicate that Gα(i/o) also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Gα(i/o) remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Gα(i3) in the GTP-bound state. The NMR spectral changes of Gα upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the K(d) value was estimated as ～1 mm. The TCS experiments identified the direct binding sites on Gα and GIRK as the α2/α3 helices on the GTPase domain of Gα and the αA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Gα transiently interacts with the αA helix of GIRK. Based on these results, we built a docking model of Gα and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Gα(i/o).     

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.