Conformational Changes Underlying Pore Dilation in the Cytoplasmic Domain of Mammalian Inward Rectifier K+ Channels

The cytoplasmic domain of inward rectifier K+ (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K+ currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.


Introduction
G protein-gated Kir channels participate in the formation of slow inhibitory postsynaptic potentials in neurons and bradycardia in the heart [1]. Four members of this channel subfamily, namely Kir3.1-Kir3.4, assemble to form a functional unit comprising a transmembrane domain (TMD) and cytoplasmic domain (CPD; Figure 1A) [2]. The direct association of the G protein βγ subunits (Gβγ) with the CPD of the channels physiologically triggers channel activation [3][4][5][6]. In addition, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which is required for the activation of all Kir channels, binds the Clinker, which connects the 2 domains [7,8], and residues in the CPD [9]. The TMD primarily functions as a gate, while the CPD receives various stimuli to control the gate. Therefore, clarifying the structural elements involved in conformational changes within the CPD can help elucidate the mechanisms that control Kir channel gating.
Crystallographic analysis suggested that dilation of the cytoplasmic pore is one of the macroscopic conformational changes in the CPD of mammalian Kir channels [8]. This is consistent with our previous study concerning the mode of binding of cations to an ion conduction pore in the CPD of Kir3.2. In particular, we determined that the pore is required to expand its inner diameter to lower the strength of the electrostatic field potential in order to facilitate ion diffusion [10]. Because cytoplasmic pore dilation also occurs in bacterial Kir channel homologs [11][12][13][14], the change in pore diameter at the CPD is assumed to be fundamental to Kir channel activity.
At the domain interface between the TMD and CPD, a membrane-facing loop between the βH and βI strands (G loop) located at the top of the cytoplasmic pore is expected to participate in allosteric domain coupling [15], and function as a cytoplasmic gate for ion conduction [16,17]. However, the loop is variably positioned with an adjacent loop between the βC and βD strands (CD loop) identified in the crystal structures in the presence [8,[18][19][20] and absence of the TMD [16,21]. Therefore, the role played by the rearrangement of structural elements within the CPD in the control of Kir channel activity remains unknown.
In this study, we focused on 2 amino acids that face the cytoplasmic pore when Kir3.2 is closed: Glu236 and Met313 ( Figure 1). These amino acids are spatially adjacent to each other, but present on neighboring β-strands [10]. We hypothesized that, if the expansion of the cytoplasmic pore is associated with G protein-dependent activation, the side chains of Glu236 and Met313 may shift position and contact their surroundings upon the transition from the closed state to the open state. Therefore, we analyzed the properties of Kir3.2 mutated at Glu236 and Met313 to gain insights into the role of structural elements within the CPD in the control of pore dilation and the relationship between structural rearrangements and the control of channel activation.

Two-electrode voltage clamp experiment
The mouse Kir3.2d isoform was subjected to functional analyses [22]. The amino acid numbers correspond to the longest mouse Kir3.2 isoform. Point mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the presence of the mutations was confirmed by direct DNA sequencing. The care of Xenopus laevis was in accordance with the guideline of the Institute of Experimental Animal Sciences, Osaka University. The protocol was approved by the Animal Experiment Committee of Osaka University. The frogs were deeply anesthetized by immersing in 0.2% tricaine methanesulfonate for 20 min at 18°C. A portion of an ovary lobe was surgically removed and incubated with collagenase to remove follicular cells. The oocytes were injected with the cRNAs of Kir3.2 and porcine m 2 -muscarinic receptor (m 2 R), and maintained in modified Barth's solution at 18°C with daily solution changes. Electrophysiological studies were undertaken 48-72 h later at room temperature (20-25°C). Two-electrode voltage-clamp recordings from oocytes were performed using a GeneClamp 500B amplifier (Axon Instruments, Sunnyvale, CA). The pipettes had a resistance of 0.5-1.5 MΩ when filled with 3 M KCl. The bath solution contained 40 mM KCl, 50 mM NaCl, 3 mM MgCl 2 , 0.15 mM niflumic acid, and 5 mM HEPES-KOH (pH 7.4). The currents were recorded in the absence or presence of various concentrations of acetylcholine (ACh). Ba 2+ (3 mM) was added at the end of the recording to estimate the endogenous leak current. The data are expressed as mean values ± standard error of the mean. For the quantitative estimation of ACh-dependent current responses of the wild-type (WT) and mutant channels, the current amplitudes at the end of test pulses at each ACh concentration ([ACh]) were fitted using the following equation: where I is the current amplitude measured at the [ACh], n H is the Hill coefficient, and EC 50 is the concentration that induces 50% of the maximal response. Min and max correspond to the minimum and maximal response, respectively; the min corresponds to zero, except in the cases of M313D and M313R. The values of n H and EC 50 were calculated using a non-linear least-squares best fit (SigmaPlot 9; Systat Software). Statistical analyses were performed by one-way ANOVA with post-hoc tests.

Patch-clamp analysis
The Kir3.2d and m 2 R subcloned into pcDNA3 (Invitrogen) were transfected into HEK293T cells using the Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). Single-channel activity was recorded in a cell-attached configuration at a holding potential of -100 mV using a patch-clamp amplifier (Axopatch 200B; Axon Instruments). The current traces were low-pass filtered at 1 kHz and digitized at 10 kHz. The glass pipettes had resistances of 2.5-3.5 MΩ when filled with the pipette solution, which contained 135 mM KCl, 1 mM CaCl 2 , 1.6 mM MgCl 2 , 5 mM HEPES, and 5 μM ACh; the bath solution was a Tyrode solution. A 50% crossing criterion for the current amplitude was used to judge the single-channel activity.
Inside-out patch clamp experiments were performed by the excision of patch membranes in the cell-attached configuration. The bath solution consisted of 135 mM KCl, 5 mM EGTA, 2 mM MgCl 2 and 5 mM HEPES. The holding potential was -100 mV. We perfused ATP at the intracellular side to produce PIP 2 at the inner leaflet of the plasma membrane, GTP (3 μM) to produce Gβγ mildly and GTPγS (10 μM) to generate Gβγ maximally [29]. The pH of all solutions was adjusted to 7.35 with KOH prior to the experiments. All experiments were conducted at ambient temperature (20-25°C).

X-ray diffraction measurements and structure analyses
The construct encoding the CPD of Kir3.2 [23] was mutated to express an arginine at position 236. The purified protein was subjected to crystallization. Crystals were grown in sitting drops by mixing 2 μL of the protein solution with 2 μL of the reservoir solution containing 15% (v/v) ethanol, 0.1 M HEPES-NaOH (pH 7.5), and 0.2 M MgCl 2 . The crystals were cryoprotected by increasing the concentration of glycerol to 25%, mounted on nylon pins, and directly immersed in liquefied nitrogen. All procedures were performed at 4°C.
The data were collected at the BL44XU beam line at the SPring-8 synchrotron radiation facility (Hyogo Prefecture, Japan), indexed and scaled using the HKL2000 suite [24]. The initial phase was determined by molecular replacement using the coordinates of the CPD of Kir3.2 (PDB code: 3AGW) [25]. The model was refined using programs in the CCP4i suite [26] and the graphics program Coot [27]. The statistics of the data collection and crystallographic analyses are presented in Table  1. Illustrations of the structures were prepared using PyMOL (DeLano Scientific LLC). The atomic coordinates and structural factors of the crystal structure of the CPD of the mouse Kir3.2 E236R mutant have been deposited in the Protein Data Bank under the accession code 3VSQ.

Western blot analysis
Kir3.2 WT and mutants were expressed in HEK293T cells. The lysate obtained from cells grown in a well of 6-well plates were subjected to SDS-PAGE and blotted on a polyvinylidene difluoride membrane. The expressed channels were detected using the anti-Kir3.2 antibody (aG2A-5) [28].

Results
In the closed state of Kir3.2, the side chains of Glu236 and Met313 are exposed to the cytoplasmic pore (Figures 1B and 1C) [8,10,23]. These amino acid residues are spatially adjacent to each other, but belong to neighboring β strands (βD and βH strands). We hypothesized that, if the expansion of the pore in the CPD is essential for channel activation, mutations at these residues may affect the property of receptor-dependent activation; even though they are not directly involved. Therefore, we tested m 2 R-dependent channel activation using proteins containing mutations at Glu236 (glycine, alanine, serine, aspartate, asparagine, valine, glutamine, and arginine) or Met313 (glycine, alanine, serine, threonine, aspartate, valine, glutamine, and arginine). In addition, we measured their current amplitudes at various membrane potentials to obtain their conductance-voltage (G-V) relationships.

Effects of mutations at Glu236 and Met313 on m 2 Rdependent activation
We measured K + currents from Xenopus oocytes injected with the cRNAs of m 2 R and either WT [22] or mutant Kir3.2. Most mutants, except E236V, exhibited Ba 2+ -sensitive K + currents ( Figure S1 in File S1). The Ba 2+ -sensitive current responses of the WT and 4 typical mutants (E236A, E236D, M313A, and M313V), induced by various concentrations of ACh, are shown in Figure 2A. The ACh-induced current was rapidly activated and slowly decreased during hyperpolarizing voltage pulses. The ACh-induced current amplitudes at the end of the test pulse at −120 mV were normalized to that recorded in the presence of 10 μM ACh ( Figures 2B and 2C). The parameters of ACh-dependent activation are summarized in Table 2. ACh activated the WT channel in a concentrationdependent manner with a Hill coefficient of 0.83 ± 0.06 and a 50% effective concentration (EC 50 ) of 35 ± 8 nM (n = 12). When Glu236 was substituted with small amino acids such as glycine (E236G) and alanine (E236A), the EC 50 values (36 ± 18 nM [n = 6] and 34 ± 5 nM [n = 11], respectively) were comparable to that of the WT. The introduction of serine (E236S), aspartate (E236D), and asparagine (E236N) increased the EC 50 to 82 ± 17 nM (n = 9), 220 ± 30 nM (n = 9), and 430 ± 80 nM (n = 7), respectively, without significantly affecting the Hill coefficient.
On the other hand, substitution to glutamine (E236Q) did not affect the EC 50 or Hill coefficient (35 ± 6 nM and 0.75 ± 0.02, respectively; n = 10). Because the E236D and E236N mutants, but not the E236Q mutant, exhibited an obvious shift in the G-V curve to the left ( Figure S2 and Table S1 in File S1), we concluded that medium-sized and non-charged amino acids efficiently prevented activation. These results suggest that Glu236 changes its position upon G protein-triggered transition from the closed state to open state, and the side chain of Glu236 associates with its surroundings. Next, we compared the receptor-dependent activation of Kir3.2 with mutations at Met313 ( Figure 2C and Figure S1 in File S1). When Met313 was substituted with glycine or alanine, the dose-response curves were similar to that of the WT; the EC 50 and Hill coefficients were 33 ± 2 nM and 0.98 ± 0.07 for . The introduction of charged residues (M313D and M313R) resulted in shallowsloped activation curves with high EC 50 values. In contrast, substitution with glutamine (M313Q) did not significantly affect receptor-dependent channel activation. These results indicate that mutations at position 313 mainly affect the slope of the activation curve. The m 2 R and G proteins mediate the AChdependent activation of the channels. This sequential reaction is thought to render the Hill coefficients derived from receptorstimulation smaller than those obtained by stimulation with Gβγ or GTP [29][30][31]. Because the difference in the current responses of mutants appears to stem from the mutants' own properties, Met313 may shift its position upon gating and associate with its surroundings.

Effects of mutations at position 313 of Kir3.2
When the expression level of the G protein-gated Kir channels was increased, the cells exhibited the K + current, even in the absence of receptor stimulation [32,33]. When this basal current amplitude (I basal ) was divided by that in the presence of 10 µM ACh (I total ) in every oocyte, the plot of the I basal /I total fraction against the I total showed a linear relationship in the WT and mutant channels when the I total was less than 10 µA (Figure 3). Assessment of the basal current fraction of the Kir3.2 WT and mutants only from oocytes with a total current of less than 2.5 μA revealed that many mutants had a higher fraction of I basal than the WT (Table 2). This suggests that the presence of Glu236 and Met313 plays a role in stabilizing the closed state, and supports the idea that the conformation of the CPD of Kir3.2 changes around the cytoplasmic pore [10]. However, because the EC 50 , Hill coefficient and I basal /I total values of the mutants varied depending on the mutated residues and introduced residues, it is not necessarily appropriate to suggest that the effects of the mutations could simply be compared.
Nevertheless, it appears that the I basal /I total values of some of the Met313 mutants are worthy of note. These mutants contained a small side chain (M313G, M313A, and M313S), exhibited EC 50 and Hill coefficient values comparable to those of the WT (Figure 2 and Table 2), and G-V relationship comparable to that of the WT ( Figure S2 in File S1). However, reductions in the volume of the side chain dramatically increased the I basal /I total ratio ( Figure 3). To reveal the mechanism underlying this phenomenon, we measured the single-channel activity of the mutants at position 313 in Kir3.2. As shown in Figure  , similar to that of the WT. This suggests that the mutation leads to the preferential existence of the open state; moreover, this property could account for the increase in the basal current level. The increased I basal may be attributed to endogenous Gβγ trafficking with newly synthesized channels to the plasma membrane [4,32,33]. The mutations in the TMD are also reported to increase the I basal [34]. To identify which mechanism, Gβγ-dependent or Gβγ-independent, contributes to the increased I basal in M313G, we expressed the Kir3.2 WT and mutant channels in HEK293T cells and measured their activity in the inside-out patch configuration ( Figure 5). After excising the patches, ATP was perfused at 2 mM to produce PIP 2 at the inner leaflet of the patch membranes. The WT was weakly activated by ATP perfusion and its activity was 15 ± 4% of that evoked by 10 μM GTPγS (n = 7). However, M313G was remarkably stimulated, by 59 ± 6% (n = 5). Taken together, the increase in the affinity to PIP 2 is likely to be responsible for the prolonged mean open time and the increased I basal of M313G. In contrast, the M313T mutant, which exhibited a reduced Hill coefficient (Figure 2), showed a mean open time (0.16 ± 0.02 ms; n = 6) that was much shorter than that of the WT ( Figure 4A). The protein expression level of M313T was comparable to that of the WT ( Figure S4 in File S1). Thus, the shortened mean open time of the single channel (Figure 4) was consistent with the observation of low current expression in HEK cells ( Figure 5) and the requirement for 5-fold more cRNA to be injected into the Xenopus oocyte to record a current amplitude comparable to that of the WT (Table 2). In contrast to M313G, M313T was less sensitive to ATP, but it was activated by GTP and exhibited about 11 ± 2% of the response evoked by GTPγS (n = 5), which was higher than the WT (4 ± 2% [n = 5]). It was difficult to identify a single-channel conductance in M313T ( Figure 4A). Because its brief dwell time in the open state might prevent the estimation of the singlechannel property, we introduced threonine at position 301 of Kir2.1, which is equivalent to Met313 of Kir3.2, and measured its single-channel activity ( Figure 4B). The mutation obviously conferred a shorter open time and unstable conduction. It was also difficult to estimate the single-channel conductance of the Kir2.1 M301T mutant. These results suggest that, even though Met313 is situated in the CPD of Kir3.2, its side-chain effectively affects the gate. Furthermore, the comparable phenotype associated with these mutants implies that, even though Kir3.2 is sensitive, whereas Kir2.1 is insensitive to Gβγ, they share a similar conformational change around the methionine on their βH strand during gating. Moreover, although M313G and M313T mutants retained the sensitivity to channel activators, they significantly reduce the sensitivity to Na + . These observations suggest that conformational change around Met313 participates in the Na + -dependent activation.

Introduction of a positive charge at position 236 in Kir3.2
Mutational analyses suggested that receptor stimulation causes a positional shift in Glu236 (Figure 2). To further explore the structural rearrangement around Glu236, we tested the effect of a reverse charge at this position by introducing arginine (E236R). Even though its protein expression level was similar to that of the WT in HEK cells ( Figure S4 in File S1), about 50-fold more E236R cRNA was required to obtain a current amplitude comparable to that of the WT ( Table 2). The mutation led to a Ba 2+ -sensitive K + current and rendered the mutant constitutively active (Figures 6A and 6B). The currentvoltage relationship of the mutant exhibited strong inward rectification (Figures 6C and 6D). Meanwhile, the mutant exhibited a shift in the G-V relationship towards the negative direction ( Figure 6E). A Kir2.1 mutant having the equivalent mutation to E236R of Kir3.2 (E224R) also conferred a rectification property similar to that of E236R on the mutant [35,36]. Next, we recorded the single-channel activity of the E236R mutant ( Figure 6F). The mutant showed rare channel openings with a single-channel conductance that was much smaller than that of the WT ( Figure 4A). The properties of a mutant in which lysine was substituted at Glu236 (E236K) was similar to that of the E236R mutant ( Figure S3 in File S1). Thus, the introduction of positive charge at position 236 appeared to yield insensitivity to Gβγ and cause a leftward shift in the G-V relationship.
Because Glu236 is present in the CPD, the characteristics of the E236R mutant could stem from the properties of the CPD. Therefore, we performed crystallographic analysis using a protein construct of the CPD of Kir3.2 containing an E236R mutation [23]. The mutant protein could be purified and crystallized under conditions similar to those of the WT (Table  1) [25]. The protein existed as a tetramer in both solution and crystals ( Figure 7A). The positions of the backbone Cα atoms in the E236R core structure were similar to those in the WT structure (root mean square deviation, 1.0 Å), suggesting that the mutant structure is comparable to a closed conformation [10]. It is noteworthy that the introduced arginine contacts the adjacent subunit via hydrogen bonding with the carbonyl oxygen atoms of Gly312 on the βH strand and Cys321 on the βI strand ( Figure 7B). The negative electrostatic field potential at the surface of the cytoplasmic pore of Kir3.2 [10,38] may facilitate the formation of inter-subunit bonds between the positively charged side chain introduced at position 236 and backbone carbonyl oxygen atoms.
These inter-subunit hydrogen bonds appear to exert 2 distinct effects on the mutant structure. The first is the immobilization of the βD strand. This strand connects to the CD loop, which shifts its position upon receptor stimulation [25,37]. The loss of pliability of the βD strand may prevent the G protein-dependent conformational change and render the E236R mutant insensitive to G proteins. The second is the immobilization of a pore-forming β sheet consisting of the βD, βH, and βI strands. The distance between the Cα atoms at site 236 in the diagonal subunits of the E236R structure was 20.1 Å, which is slightly wider than that in the closed state of the WT structure (16.5 Å). This feature may be insufficient to prevent K + passage, even though it possesses a strong negative electrostatic potential [10,38]. Cys321 of Kir3.2 is accessible to sulfhydryl modifiers from the cytoplasmic side [37]. The position tends to adapt to the amino acid substitution and the property of the parent dominates those of the mutants ( Figure S3 in File S1) [37]. On the other hand, although neither the G312A nor the G312Y mutation on the E236R mutant was functional, the corresponding mutant of Kir2.1 (G300A) retained the WT property [16]. Therefore, the introduction of arginine at position 236 may affect the rigidity of the pore-forming β sheet and constrain the full opening of the cytoplasmic pore, rather than deteriorate the domain coupling mediated by the side chains of Gly312 and Cys321. Taken together, these inter-subunit bonds may contribute to the unique conduction of the E236R mutant.

Discussion
The Kir channel is a ligand-operated membrane protein, and its CPD receives various stimuli to control the gate at the TMD. In this study, we analyzed conformational changes around the cytoplasmic pore in the CPD of Kir3.2 by focusing on the porefacing residues Glu236 and Met313. Even though these 2 residues are spatially adjacent to each other in the closed conformation, the effects of mutagenesis on receptordependent activation differed between them. In particular, the Glu236 mutants had increased EC 50 values and the Met313 mutants had decreased Hill coefficient values ( Figure 2). Furthermore, the inter-subunit connections generated in the E236R mutant appear to result in a leftward shift in the G-V relationship and G protein insensitivity (Figures 6 and 7). These results indicate that, when Kir3.2 functions, the CPD substantially alters its conformation. In particular, the region around the cytoplasmic pore is altered and the most likely change is associated with the expansion of the pore.
In the crystal structures of Kir3.2, Glu236 protrudes into the cytoplasmic pore ( Figure 1). This conformation is supported by the disruption of the β-type hydrogen bonds between the amino acids at 236 and 237 on the βD strand, and a residue at 312 on the βH strand ( Figure 7C). This structural feature is known as a β-bulge [39], which is thought to confer structural flexibility to the β sheet. The β-bulge is also observed at the corresponding regions of Kir3.1 and Kir2.1 [16], and Kir3.1 exhibits significant displacement of the backbone at a residue corresponding to Glu236 of Kir3.2 and the connecting CD loop in 2 different crystal forms [16,21]. We previously reported that interference of the conformational change of the CD loop by Cd 2+ [37] or its N-terminus [25] hampers the activation of Kir3.2. The displacement of the CD loop is expected to be coupled to PIP 2binding [25,40]. Therefore, the G protein might allow the channel to bind PIP 2 and then shift the position of the βD strand and the connecting CD loop as a unit.
The βH strand, which harbors Met313, is also expected to shift its position during gating (Figures 2, 3, 4, and 5, and Figures S1 and S2 in File S1). The βH strand associates with the βI strand by β-type hydrogen bonds and specifically contacts the arginine introduced at position 236 of the adjacent subunit in the E236R mutant ( Figure 7). Thus, it could be anticipated that the βH strand forms an element with the βI strand and the structurally flexible G loop that is sandwiched by the 2 strands. The introduction of threonine at Met313 in G protein-sensitive Kir3.2 and at the corresponding residue in G protein-insensitive Kir2.1 notably resulted in the same g / g max = g min + 1−g min / 1+exp V −V 1/2 / k where V 1/2 is the half-maximal activation voltage and k is the slope factor: WT: V 1/2 = −34 ± 4 mV, k = 27 ± 3 mV (n = 10); E236R: V 1/2 = −78 ± 1 mV, k = 28 ± 1 mV (n = 11). Error bars indicate the standard error of the mean. All error bars are smaller than the symbols used.  phenotype: short open dwell time and unstable conductance at the single-channel level (Figure 4). Because the mutations at Met313 potentially disturb the conformation of the G loop, these findings are insufficient to exclude the possibility that the G loop functions as a cytoplasmic gate [16,17]. Nevertheless, these results suggest that the role of the βH strand and its surroundings in the CPD in the regulation of gating is similar in Kir channels despite the sensitivity to G proteins. The most recent crystal structure of Kir3.2 suggests that the G loop is sandwiched by 2 TM2s of adjacent subunits [20]. Therefore, it might be possible that the element accommodating the βH strand concomitantly moves with the TMD at each step of single-channel gating, even though the element is part of the CPD.
So far, the G protein-gated Kir channels have been proposed to exhibit rotational motion of the CPD against the TMD during gating [41]. Based on the crystal structure of mammalian Kir3.2 in complex with PIP 2 , Na + and Gβγ [20], it is proposed that the interaction of Gβγ at the interface of the subunit in the CTD evokes the twist against the TMD without disturbing the conformation of the CTD. The manner of this CTD-originated rotational motion of the channel is in sharp contrast with the motion observed in KcsA, in which its TMD twists during gating by itself [42]. A ligand-induced conformational change at the ligand-binding domain is thought to mechanically force the opening of the activation gate at the TMD in ligand-operated ion channels such as ionotropic glutamate receptors [43,44] and Ca 2+ -activated K + channels [45][46][47]. Even though the pore-forming βD and βH strands contribute to the dilation of the cytoplasmic pore, they seem not to move as a combined unit. This β-bulge-based structural feature of the βD and βH strands raises the question of whether the conformational change in the βD strand generates the tension that drives the TMD by regulating the position of the βH strand. Furthermore, how such microscopic rearrangements of these structural elements couple to the macroscopic conformational changes observed at the domain level should be clarified in the near future. Further structural data on the open channel state combined with mutational analyses are required to elucidate the functional implications of pore dilation in the CPD and the coupling between the 2 domains in the regulation of Kir channel gating.