Tuning the ion selectivity of two-pore channels - PNAS

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Jan 31, 2017 - Jiangtao Guoa, Weizhong Zenga,b, and Youxing Jianga,b,1. aDepartment of Physiology ...... EMBO J 34(13):1743–1758. 27. Wang X, et al.
Tuning the ion selectivity of two-pore channels Jiangtao Guoa, Weizhong Zenga,b, and Youxing Jianga,b,1 a Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040; and bHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040

Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved December 16, 2016 (received for review September 28, 2016)

two-pore channel

| ion selectivity | crystal structure

T

wo-pore channels (TPCs) are organellar cation channels ubiquitously expressed in animals and plants (1, 2) and belong to the voltage-gated ion channel superfamily (3). TPC channels contain two homologous Shaker-like six-transmembrane (6-TM) domains in each subunit and function as homodimers. They are believed to be evolutionary intermediates between homotetrameric voltage-gated potassium channels and the four-domain, single-subunit, voltage-gated sodium/calcium channels (4). In human and animals, TPC channels (TPC1 and TPC2) are localized to the endo/lysosomal membranes and regulate the ionic homeostasis within these acidic organelles. Their functions have been shown to be involved in various physiological processes, such as endocytosis and endosomal trafficking (5, 6), lysosomal morphology and pigmentation (7, 8), autophagy (9, 10), and nutrient metabolism via the mammalian target of rapamycin (mTOR) complex (11). Not surprisingly, given their central physiological role, defects in TPC channels are associated with a variety of disabling human disorders (9, 12–17). Despite their physiological importance, some biophysical properties of mammalian TPC channels are still under debate (18). Mammalian TPC channels were initially identified as receptors of nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+mobilizing second messenger, and responsible for Ca2+ release from the acidic organelles (19–21). Although the NAADP-dependent endo/lysosomal Ca2+ release is attributed directly to the Ca2+ conduction of TPC channels activated by NAADP in some studies (16, 22–26), several recent studies demonstrate that TPCs are Na+selective channels that are activated by phosphatidylinositol 3,5bisphosphate [PI(3,5)P2], instead of NAADP (11, 27), however. The Na+ permeability of TPCs was also reported in several other recent studies (26, 28, 29). In this study, we aim to provide structural and functional insights into the ion selectivity properties of TPC channels by taking advantage of our recently reported crystal structure of a plant TPC1 from Arabidopsis thaliana, AtTPC1, which is a voltagegated, Ca2+-regulated, nonselective cation channel localized to the vacuolar membrane (30). Despite the difference in ion selectivity www.pnas.org/cgi/doi/10.1073/pnas.1616191114

between AtTPC1 and its human counterparts, their filter sequences are quite conserved, particularly between AtTPC1 and human TPC2 (HsTPC2), indicating a similar overall structure at the selectivity filter region. Here, we performed electrophysiological characterization and comparison of the ion selectivity properties of both AtTPC1 and HsTPC2. Through structure-guided mutagenesis, we were able to convert the nonselective AtTPC1 to a Na+-selective channel similar to HsTPC2, and thereby identified the key filter residues that are central to defining TPC selectivity. We also determined the structure of the Na+-selective AtTPC1 mutant, which, in comparison to AtTPC1, provides structural insights into the selectivity mechanism in TPC channels. Results Ion Selectivity of AtTPC1. We overexpressed AtTPC1 in HEK293

cells (Fig. S1) and measured the channel activity on the plasma membrane using patch clamping in the whole-cell configuration (Materials and Methods). Under this setting, the extracellular side (bath side) is equivalent to the luminal side of AtTPC1 in vacuoles. To determine the selectivity of AtTPC1, the membrane potential was stepped to +80 mV for channel activation and then switched to various testing potentials. The tail currents were recorded to generate a current-voltage (I-V) curve for determination of the reversal potential. The standard pipette solutions (cytosolic side) contained 150 mM Na+; therefore, all selectivity measurements of AtTPC1 among various monovalent and divalent cations were compared with Na+. Under bi-ionic conditions with 150 mM Li+, Na+, or K+ in the bath solutions, the reversal potentials were all close to 0 mV, indicating that AtTPC1 exhibits almost equal permeability to these ions (Fig. 1 A, B, and D). AtTPC1 is less selective for Rb+ and Cs+, with reversal potentials of −20.4 ± 4.6 mV and −39.6 ± 3.3 mV, respectively, yielding a permeability (P) ratio PNa/PRb of 2.2 and a Significance Ion channels selectively transfer ions across cell membranes, and their selectivity is controlled by a special region of the channel protein called the selectivity filter. Two-pore channels (TPCs) belong to the superfamily of voltage-gated tetrameric cation channels and possess a unique set of filter residues that define their ion selectivity. Despite extensive studies, debate still lingers about the selectivity properties of mammalian TPCs. Here, we provide structural and functional insights into the selectivity properties of TPC channels. We confirm the Na+ selectivity of human TPC2, identify the key residues in the TPC filters that differentiate the selectivity between mammalian TPC2 and plant TPC1, and reveal the structural basis of Na+ selectivity in mammalian TPCs. Author contributions: J.G., W.Z., and Y.J. designed research; J.G. and W.Z. performed research; J.G., W.Z., and Y.J. analyzed data; and J.G., W.Z., and Y.J. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 5TUA). 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1616191114/-/DCSupplemental.

PNAS | January 31, 2017 | vol. 114 | no. 5 | 1009–1014

BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Organellar two-pore channels (TPCs) contain two copies of a Shakerlike six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region, yet exhibit drastically different ion selectivity. Plant TPC1 functions as a nonselective cation channel on the vacuole membrane, whereas mammalian TPC channels have been shown to be endo/lysosomal Na+-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity with the selectivity of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is selective for Ca2+ over Na+, but nonselective among monovalent cations (Li+, Na+, and K+). Our results also confirm that HsTPC2 is a Na+-selective channel activated by phosphatidylinositol 3,5-bisphosphate. Guided by our recent structure of AtTPC1, we converted AtTPC1 to a Na+-selective channel by mimicking the selectivity filter of HsTPC2 and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na+-selective AtTPC1 mutant elucidates the structural basis for Na+ selectivity in mammalian TPCs.

PNa/PCs of 4.7. Because AtTPC1 is known to conduct Ca2+, we also measured the relative selectivity between Ca2+ and Na+. Because luminal Ca2+ can inhibit AtTPC1 activity by shifting voltage activation toward a more positive potential (30, 31), we generated a mutant channel, AtTPC1ΔCai, which contains three mutations (Asp240Ala, Asp454Ala, and Glu528Ala) at the inhibition site; these mutations mitigate the Ca2+ inhibition but have no effect on ion permeation (Fig. S2), allowing for the measurement of the Ca2+ current. As shown in Fig. 1C, with the presence of 15 mM Ca2+ in the bath solution and 150 mM Na+ in the pipette, AtTPC1ΔCai has a reversal potential of about 0 mV, indicating a higher selectivity for Ca2+ over Na+ with a PCa/PNa of about 5 (Fig. 1 C and D).

A

B

Na+ K+ Li+

Na+150 X+ 150

80 mV

40 mV

-120

1.5 1.0

Converting AtTPC1 to a Na+-Selective Channel. Based on the structure

1

-40

0

-70 mV Current (nA)

-120 mV control cell

40 Vm (mV)

-1

-2

+

1 nA

Na 150

C

200 ms

Na+150 Ca2+ 15

60 mV

100 mV

-70 mV

-100 mV

Li+ 150

200 ms Current (nA)

1 nA

Ca2+ 15

+

K 150

0.5

Ca2+

0 -100

-50

0

50 Vm (mV)

-0.5

Rb+ 150

Cs+ 150

D ion

Erev (mV)

PNa/PX

Na+

0±2.8

1.0

Li+

0±3.7

1.0

K+

0±4.2

1.0

Rb+

-20.4±4.6

2.2

Cs+

-39.6±3.3

4.7

Ca2+

0±3.7

0.2

Fig. 1. Ion selectivity of AtTPC1. (A) Sample traces of whole-cell currents of AtTPC1 overexpressed on the plasma membrane of HEK293 cells with 150 mM Na+ in the pipette solution and 150 mM X+ (X = Li, Na, K, Rb, or Cs) in the bath solution. All recordings shown here were performed on a single patch, and the bath solutions were exchanged through perfusion. (B) I-V curves generated from the tail currents of the traces shown in A. (C) Sample traces of whole-cell currents with 150 mM Na+ in the pipette solution and 15 mM Ca2+ in the bath solution and the corresponding I-V curve. (D) Summary of reversal potentials of AtTPC1 with various cations in the bath solutions and the calculated relative permeability of these ions in comparison to Na+. The reversal potential values are the mean ± SEM of at least five measurements from different patches. Vm, membrane potential.

1010 | www.pnas.org/cgi/doi/10.1073/pnas.1616191114

HsTPC2 Is a Na+-Selective Channel. Contrary to plant TPC1, mam-

malian TPC channels have been shown to be selective, despite a disagreement on whether they are selective for Na+ over Ca2+. To compare the selectivity properties between plant and mammalian TPCs, we chose HsTPC2 as the model system because it has a high sequence similarity to AtTPC1 at the filter region (Fig. 2A). By replacing two leucines with alanines (L11A/L12A) on the N-terminal lysosomal targeting sequence, HsTPC2 can be overexpressed and trafficked to the plasma membrane in HEK293 cells (Fig. S1), allowing for direct measurement of channel activity by patching the plasma membrane (22, 28). We applied this strategy and recorded channel activity of HsTPC2 using the inside-out patch configuration (Materials and Methods). In our recordings, HsTPC2 on the plasma membrane was activated by cytosolic PI(3,5)P2 (10 μM in bath), but no obvious channel activation by NAADP in a wide range of concentrations (10 nM ∼ 10 μM) was observed (Fig. 2B). Under biionic conditions with 150 mM Na+ in the pipette and 150 mM K+ in the bath solution, HsTPC2 has a reversal potential of about 81 mV, indicating high selectivity for Na+ with a PNa/PK of 23.8 (Fig. 2B). To measure the selectivity between Ca2+ and Na+ or K+ for HsTPC2, we included 100 mM Ca2+ in the pipette (extracellular) and 150 mM Na+ or K+ in the bath solution. In this setting, the channel shows high selectivity for Na+ over Ca2+ with a reversal potential of about −50 mV, yielding a PNa/PCa of about 16.8; the channel shows no selectivity between K+ and Ca2+ with a reversal potential of about 7 mV, yielding a PCa/PK of about 1.2 (Fig. 2C). Therefore, our study demonstrates that HsTPC2 is a Na+-selective channel activated by PI(3,5)P2. Our results are consistent with the observations from whole-lysosome recordings of PI(3,5)P2-activated mammalian TPC2 in enlarged lysosomes (11, 27), but different from whole-lysosome planar patch-clamp recordings of NAADPactivated currents (24, 26). Albeit small, we did observe inward Ca2+ current in our recording. Considering the large [Ca2+] gradient (∼10,000-fold) across the lysosomal membrane, it is possible that a small fraction of HsTPC2 current could come from Ca2+ in vivo.

Rb+ Cs+

-80

Therefore, under our recording conditions, AtTPC1 is permeable to various cations and has a relative permeability sequence of Ca2+ > Na+ ∼ Li+ ∼ K+ > Rb+ > Cs+ (Fig. 1D).

of AtTPC1 and the filter sequence alignment, the major differences between AtTPC1 and HsTPC2 lie in the three central filter residues that form the ion pathway (Fig. 2A). AtTPC1 has a sequence of 264TSN266 at filter I (from the first 6-TM domain) and 629MGN631 at filter II (from the second 6-TM domain), whereas HsTPC2 has 271TAN273 and 652VNN654, respectively. To test if we can recapitulate the selectivity property of HsTPC2 in AtTPC1, we replaced the filter residues of AtTPC1 with the filter residues of HsTPC2 (i.e., a triple mutant with S265A in filter I and M629V/ G630N in filter II). We named the mutant channel At2HsTPC2 and performed the same selectivity measurement as for the wildtype AtTPC1. These filter mutations do not change the gating of AtTPC1 (Fig. S3) but have a profound effect on channel selectivity. As shown in Fig. 3 A and B, although At2HsTPC2 is still equally permeable to Li+ and Na+, the channel becomes highly selective for Na+ over larger monovalent cations (K+, Rb+, and Cs+). With K+ in the bath, the reversal potential is −95.4 ± 5.8 mV, yielding a PNa/PK of about 41. To compare the selectivity between Na+ and Ca2+, we generated the At2HsTPC2 mutant (At2HsTPC2ΔCai) on the background of AtTPC1ΔCai to mitigate extracellular Ca2+ inhibition. With 15 mM Ca2+ in the bath solution and 150 mM Na+ in the pipette, At2HsTPC2ΔCai has a reversal potential of about −50 mV, indicating a higher permeability for Na+, with a PNa/PCa of about 2.5 (Fig. 3 C and D). Thus, by mimicking the HsTPC2 filters, we are able to recapitulate the ion selectivity of HsTPC2 and convert AtTPC1 to a Na+-selective Guo et al.

IP1 filter I IP2

AtTPC1 HsTPC2 HsTPC1

ILFTTSNNPDV 271 VLLTTANNPDV 277 VLLTTANFPDV 285

B

C Na+ 150

-100

0

-50 Vm (mV)

50

Na Na+ or K+ 150

K+ 150

Current (nA)

IIP1 filter II IIP2

NLLVMGNWQVW 635 NLMVVNNWQVF 658 ELTVVNNWYII 652

+

1.0

K+

Control

-1

AtTPC1 HsTPC2 HsTPC1

1.5

Ca2+ 100

100

Current (nA)

A

0.5

NAADP 30 nM PI(3,5)P2 10 µM -2

-100

-50

0

50 Vm (mV)

100

Fig. 2. Ion selectivity of HsTPC2. (A) Partial sequence alignment of AtTPC1, HsTPC1, and HsTPC2 at the filter regions. IP1, filter I, and IP2 mark the pore helices and selectivity filter from the first 6-TM domain; IIP1, filter II, and IIP2 are from the second 6-TM domain. The three central filter residues are shown in red. (B) I-V curves of HsTPC2 in the presence of cytosolic PI(3,5)P2 or NAADP. A wide range of NAADP concentrations (10, 30, and 100 nM and 1 and 10 μM) was tested, and all gave the similar results. Therefore, only a representative trace with 30 nM NAADP is shown. The pipette solution contains 150 mM Na+, and the bath solution contains 150 mM K+. (C) I-V curve of HsTPC2 activated by 10 μM PI(3,5)P2, with 100 mM Ca2+ in the pipette solution and 150 mM Na+ or K+ in the bath solution. All currents were recorded using inside-out patches. The measured reversal potentials and calculated relative permeability values between Na+ and K+ or Ca2+ are listed in Table 1.

Residues on Filter II Define the Selectivity of TPC Channels. To test whether all three filter mutations in At2HsTPC2 are necessary for achieving high Na+ selectivity, we also measured the selectivity of AtTPC1 with single and double mutations in the filter under bi-ionic conditions with 150 mM Na+ in the pipette and 150 mM K+ in the bath solution (Fig. S4). As summarized in Table 1, none of the single mutations can change the selectivity of AtTPC1; a double mutation in filter II, M629V/G630N, is necessary to convert AtTPC1 to a Na+selective channel; S265A does not play a determinant role but can further enhance the Na+ selectivity of the channel. Thus, having Val and Asn together in filter II appears to be essential for Na+ selectivity in mammalian TPCs. To cross-validate this finding, we performed reversed mutagenesis analysis on HsTPC2 by swapping the three equivalent filter residues with the filter residues of AtTPC1, either individually or collectively (Fig. S5 and Table 1). Our results show that the A272S mutation in filter I has a subtle effect on the Na+ selectivity of HsTPC2, consistent with the Na+ selectivity of mouse TPC2, which has a sequence of 255TSN257 at filter I (27). Any mutations involving Val651 and Asn652, whether single or double, can significantly decrease the selectivity of the channel, and the triple mutant with the filter sequence equivalent to AtTPC1 has the lowest Na+ selectivity. Although we were not able to abolish Na+ selectivity in HsTPC2 completely, our mutagenesis results are qualitatively consistent with the results observed in the AtTPC1 mutants, confirming the necessity of having the Val/Asn pair in filter II to achieve

high Na+ selectivity in HsTPC2. Furthermore, the loss of Na+ selectivity in the HsTPC2 triple mutant is also accompanied by more than a 10-fold increase in Ca2+ permeability with a PNa/PCa of about 1.3, similar to what was observed in our AtTPC1 study (Fig. S5B and Table 1). Structural Comparison Between AtTPC1 and At2HsTPC2. At2HsTPC2 exhibits similar ion selectivity properties as HsTPC2, providing us with a valid model system to reveal the structural basis that differentiates the selectivity between AtTPC1 and HsTPC2. We therefore crystallized the At2HsTPC2 channel and determined its structure by molecular replacement using the wild-type AtTPC1 as the search model (30, 32). The structure of At2HsTPC2 is virtually identical to the structure of AtTPC1 except in the filter region, which contains the three mutations. No obvious main chain conformational changes were observed between the wild-type and mutant AtTPC1, and all structural differences reflect changes in side-chain size at the mutated residues. The ion permeation pathway of AtTPC1 is asymmetrical at the filter region (Fig. 4 A and B). Its cross-section along the filter I pair is shorter but wider, with a minimum atom-to-atom distance of about 9 Å (Fig. 4C). The loss of the hydroxyl group, resulting from the S265A mutation, has little impact on the diagonal dimension between the filter I pair and, as expected, has little effect on channel selectivity. The filter II residues, on the other hand, build a longer but narrower part of the ion conduction pathway with a 4.8-Å-wide constriction point formed by the side chains of Asn631 from each subunit of the dimer. The G630N mutation adds another layer of

Table 1. Ion permeability of Na+, K+, and Ca2+ in AtTPC1 and HsTPC2 and their filter mutants AtTPC1 Naint(150)/ Kext(150)

Filter sequence I

II

TSN MGN TAN MGN TAN MNN TAN VGN TSN VGN TSN MNN TSN VNN TAN VNN

HsTPC2

Construct

Erev, mV

WT 0± S265A −8.1 ± — — M629V 0± G630N 0± M629V/G630N −74.4 ± S265A/M629V/ −95.4 ± G630N(At2HsTPC2)

Naint(150)/ Caext(15)*

PNa/PK

2.8 3.1

1.0 1.4

2.1 3.2 4.2 5.8

1.0 1.0 18.0 41.2

Erev, mV

Naext(150)/ Kint(150)

PNa/PCa

Construct

0 ± 3.7

0.2

−50.3 ± 6.5

2.5

A272S/V652M/N653G V652M/N653G V652M N653G — — A272S WT

Erev, mV

PNa/PK

± ± ± ±

3.2 4.1 3.7 3.3

2.9 5.1 6.8 6.5

75.3 ± 5.1 81.4 ± 4.9

18.8 23.8

27.1 42.0 49.2 48.0

Naint(150)/ Caext(100) Erev, mV

PNa/PCa

0 ± 2.4

1.3

50.6 ± 3.8

16.8

Erev, reverse potential; ext, extracellular; int, intracellular; WT, wild type. *To mitigate extracellular Ca2+ inhibition, constructs of AtTPC1ΔCai and At2HsTPC2ΔCai were used for PNa/PCa measurements.

Guo et al.

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channel. The gain of Na+ selectivity in the AtTPC1 mutant is also accompanied by the loss of its Ca2+ selectivity.

A

B Na+ 150 X+ 150

-70 mV

80 mV

Rb+ Cs+

Na+ K+ Li+

40 mV

3 2 1 0

-120

-80

0

-40

Current (nA)

-120 mV

-1

40

Vm (mV)

-2 -3 -4

1 nA

Na+ 150

C Na+ 150 Ca2+ 15

200 ms

60 mV

100 mV -70 mV

-100 mV

Li+ 150

200 ms

Current (nA)

0.5 nA

Ca2+ 15

Ca2+

1.0 0.8 0.6 0.4 0.2 0

K+ 150

0

-50

-100

-0.2

50

Vm (mV)

D ion

Rb+ 150

Cs+ 150

Na Li+

Erev (mV) +

0±4.2 0±3.8

PNa/PX 1.0 1.0

K+

-95.4±5.8

41.1

Rb+

-98.7±6.5

46.7

Cs+

-101.0±8.8

51.1

Ca2+

-50.3±6.5

2.5

Fig. 3. Ion selectivity of At2HsTPC2. (A) Sample traces of whole-cell currents of At2HsTPC2 overexpressed in HEK293 cells with 150 mM Na+ in the pipette solution and 150 mM X+ (X = Li, Na, K, Rb, or Cs) in the bath solution. All recordings shown here were performed on a single patch. (B) I-V curves generated from the tail currents of the traces shown in A. (C) Sample traces of At2HsTPC2 currents with 150 mM Na+ in the pipette solution and 15 mM Ca2+ in the bath solution and the corresponding I-V curve. (D) Statistics of At2HsTPC2 reversal potentials (Erev) measured in various bi-ionic conditions and the calculated relative permeability between Na+ and other cations. The reversal potential values are the mean ± SEM (n ≥ 5).

constriction along the pathway just below Asn631, with a similar diagonal distance of about 4.8 Å. The Asn630 side chain in the At2HsTPC2 mutant is stabilized by hydrogen bonds with the backbone carbonyls of Thr264 and Ala265 (Fig. 4B). Centered between the twofold-related Asn630 residues is an electron density peak that likely comes from a Na+ ion or a water molecule. Because the distance between the side-chain carbonyl oxygen of Asn630 is too short for a hydrogen bond to a water molecule, about 2.4 Å, we assigned this central density peak as a Na+ ion (Fig. 4B). Due to the resolution limit, we did not observe any water molecules nearby. However, considering the coordination number and ligand chemistry commonly seen for Na+, this bound Na+ ion has to be partially hydrated. The two neighboring layers of Asn residues in the At2HsTPC2 filter also create a trapping site for a larger monovalent cation in between. As shown in Fig. 4D, structures from crystals soaked with Rb+ or Cs+ revealed a strong electron density peak with anomalous 1012 | www.pnas.org/cgi/doi/10.1073/pnas.1616191114

scattering sandwiched between the Asn630 and Asn631 residues, indicating a bound Rb+ or Cs+ ion. The bound Rb+ or Cs+ in the filter likely serves as a permeating blocker and reduces the Na+ current in At2HsTPC2. No equivalent Rb+ or Cs+ binding was observed in the wild-type AtTPC1. The M629V mutation does not appear to have a direct impact on the size of the ion pathway. However, replacing the larger side chain of Met629 with the side chain of Val vacates a space beside Tyr656 from the S6 helix of the second 6-TM domain (IIS6), allowing the Tyr656 side chain to move closer to the filter. Consequently, Tyr656 in the M629V mutant engages in new packing interactions with two highly conserved filter residues: Its hydroxyl group forms a hydrogen bond with the Thr263 side chain, and its aromatic ring forms a π-stacking interaction with the aromatic ring of Trp632 (Fig. 4E). These newly formed interactions likely increase the stability of the filter. Discussion In this study, we were able to convert the nonselective AtTPC1 to a Na+-selective HsTPC2-like channel; specifically, we show that residues on filter II contribute to the difference in ion selectivity between plant and mammalian TPC channels. Structural comparison between the wild-type AtTPC1 and its Na+-selective mutant allowed us to propose a simple model to explain the structural mechanism underlining the selectivity difference. Because the At2HsTPC2 filter has an asymmetrical ion pathway with a wider dimension between the filter I pair but narrower between the filter II pair, the permeating ions likely pass through the filter in a partially hydrated form, particularly when crossing the two constriction points formed by the two pairs of symmetry-related asparagine residues (Asn630s and Asn631s). These asparagine side chains (likely the carbonyl oxygen atoms) can participate in ion coordination and stabilize the partially hydrated permeating ions. Commonly seen ion–oxygen coordination tends to have an optimal distance of about 2.4 Å for Na+ and 2.8 Å for K+ (33). With a distance of about 4.8 Å between the two asparagine residues at each constriction point, their side-chain carbonyl oxygen could provide optimal coordination to a central Na+ ion with a distance of about 2.4 Å, but too close for K+ or larger ions. In other words, by participating in ion coordination, these asparagine residues can function as a size sieve to sift out larger cations. Interestingly, when Asn631 is replaced by alanine in At2HsTPC2, the channel remains highly selective for Na+ over K+ (Fig. S6), suggesting that the two layers of asparagine residues in the At2HsTPC2 filter are not equivalent in defining Na+ selectivity, with the Asn630 residues playing the determinant role. One explanation could be the environmental difference between Asn630 and Asn631. The Asn631 side chain forms the external entrance of the filter and, without spatial confinement, its side-chain carboxamide has the freedom to rotate away from the central axis, making the Asn631 constriction point more permissive for ions of different sizes. The Asn630 side chain, on the other hand, is located in the middle of the filter with a confined space and is stabilized by hydrogen-bonding interactions with nearby filter I residues. Thus, the carboxamide groups of the two symmetrical Asn630 residues are in a defined position with less mobility, allowing them to exert stringent size selection for the crossing ions. Contrary to the G630N mutation, the M629V mutation does not appear to contribute to a size change in the filter, which raises the question about the necessity of having both the M629V and G630N to achieve high Na+ selectivity. The major effect of the M629V mutation is the formation of new protein packing interactions between Tyr656 on the IIS6 helix and two filter residues. We suspect that the newly formed protein packing surrounding the filter adds extra stability to the filter conformation in the mutant and constrains the dynamic motion of the filter, making it more restrictive for ion size. The necessity of having a stable filter to achieve high selectivity has also been observed in our previous study on K+ channels, in which weakening Guo et al.

A

B W632

IIS6

N631

IIP2 IIS5

N630

N266 N631 Na

V629 Na

IIP1

N630

IP2

T264

IS5

T263

T264

At2HsTPC2 pore domain

N266

D N266 N267 9.0 Å S265/A265 T263 T264

distance along pore axis (Å)

C

filter I

N630

35 AtTPC1 At2HsTPC2 30 1 2 3 4 Pore radius (Å)

5

E

W632 G630/N630 V628 M629/V629

distance along pore axis (Å)

N631

4.8 Å

IIP2 selectivity filter

45 4.8 Å

N631

40

0

filter II

V629

N267

T263

N631

IP1

W632

40 G630

N630

T263

35

π IIS6

AtTPC1 At2HsTPC2

M629/V629

30 0

1

2 3 4 Pore radius (Å)

5

Y656

Fig. 4. Selectivity filter structure of At2HsTPC2. (A) Structure of the At2HsTPC2 ion conduction pore in top view. The pore helix and filter regions are colored in green and salmon, respectively. The putative sodium ion in the filter is shown as a purple sphere. (B) Zoom-in view of the ion selectivity region from the top (Left) and side (Right), with the front filter I removed for clarity. The surface-rendered model is calculated without Asn630 and Asn631 side chains to reveal the dimensions of the ion pathway in the absence of the two constriction points. The electron density (light blue mesh, contoured to 4σ) of the bound Na+ is calculated from the 2Fo-Fc map. Yellow dashed lines indicate H-bonds between N630 and neighboring backbone carbonyls from filter I and the ion coordination between N630 and Na+. (C) Superimposition of the filter structures between AtTPC1 (gray) and At2HsTPC2 (filter I in green and filter II in salmon). The pore radius diagrams for the cross-sections of filter I (Top) and filter II (Bottom) from both channels are calculated with the program HOLE (35). (D) Anomalous difference maps reveal the binding of Rb (blue mesh, contoured to 5σ) or Cs (magenta mesh, contoured to 5σ) in the At2HsTPC2 filter. (E) Local structural changes caused by the M629V mutation. Residues involved in structural changes are shown in blue for AtTPC1 and in green (filter I) and salmon (filter II) for At2HsTPC2. The yellow arrow indicates the ion pathway along the channel pore.

or disrupting protein packing interactions surrounding the filter can destabilize the filter conformation and compromise the channel’s selectivity (34). Materials and Methods The X-ray diffraction data collection and structure refinement statistics are listed in Table S1. The atomic coordinates and structure factors have been deposited in the Protein Data Bank under ID code 5TUA. Detailed methods of protein purification, crystallization, structure determination, and electrophysiology are provided in SI Materials and Methods. ACKNOWLEDGMENTS. We thank N. Nguyen for manuscript preparation, J. Liou and W. Lee for confocal imaging, and D. Ren (University of Pennsylvania)

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and S. Muallem (NIH) for providing clones of AtTPC1 and HsTPC2 for functional assay. The experimental results reported in this article derive from work performed at Argonne National Laboratory, GM/CA (23ID) at the Advanced Photon Source, and at the Berkeley Center for Structural Biology at the Advanced Light Source (ALS). The Argonne National Laboratory is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under Contract DEAC02-06CH11357. The Berkeley Center for Structural Biology is supported, in part, by the NIH, National Institute of General Medical Sciences, and Howard Hughes Medical Institute. The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract DE-AC02-05CH11231. This work was supported, in part, by the Howard Hughes Medical Institute and by grants from the NIH (Grant GM079179 to Y.J.) and the Welch Foundation (Grant I-1578 to Y.J.).

PNAS | January 31, 2017 | vol. 114 | no. 5 | 1013

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IP1

V628

90o

V628

A265

IS6

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