Voltage-Dependent Gating Underlies Loss of ENaC Function in Pseudohypoaldosteronism Type 1

Transcription

1 193 iophysial Journal Volume pril Voltage-Dependent Gating Underlies Loss of ENa Funtion in Pseudohypoaldosteronism Type 1 Volodymyr Kuher, Nina oiko, Oleh Pohynyuk, and James D. Stokand * Department of Physiology, University of Texas Health Siene enter at San ntonio, San ntonio, Texas; and Department of Integrative iology and Pharmaology University of Texas Health Siene enter at Houston, Houston, Texas STRT Here we explore the mehanism and assoiated struture-funtion impliations of loss of funtion for epithelial Na þ hannel (ENa) ontaining a pseudohypoaldosteronism type 1 (PH-1)-ausing missense point mutation. s expeted, human ENa that ontained subunits harboring PH-1-ausing substitutions within an absolutely onserved, ytosoli Gly residue (e.g., bg37s) had signifiantly less ativity. Unexpetedly, though, suh substitution also results in voltage sensitivity with greater ativity at hyperpolarizing potentials. This is a onsequene of voltage-dependent hanges in the single-hannel open probability and is not speies- or subunit-dependent. Voltage sensitivity in PH-1 mutants stems from the disruption of ritial struture, rather than the development of new properties resulting from the introdution of novel side hains. Residues near the onserved His-Gly sequene of G9 in a-mena are partiularly important for voltage sensing. lthough substitution of I93 in a-mena results in voltage sensing, it also slows the ativation and deativation kinetis enough to enable apture of the dynami hanges in single-hannel open probability that aount for hanges in marosopi ativity. This provides definitive proof of the mehanism that underlies loss of funtion. In addition, the voltage dependene of ENa with PH-1 substitutions is akin to that whih results from substitution of a ritial, interfaial Trp residue onserved at the intraellular base of TM1 (e.g., W112 in a-mena). Dynami interations between similarly positioned His and Trp residues are essential for gating and the girdle-like struture that lines the intraellular mouth of the M2 proton hannel. The similar residues in ENa may serve a shared funtion, suggesting the possibility of an intraellular girdle just below the mouth of the ENa pore. INTRODUTION The epithelial Na þ hannel (ENa) is a member of the ENa/Degenerin ion hannel family (1 3). id-sensing ion hannels (SIs) of entral and peripheral neurons involved in touh and pain sensation are also members of this hannel family (4 6). ENa is ommon to the apial membrane of reabsorptive epithelial ells. It is seletive for ations over anions and greatly favors Na þ over other ations. ENa gates in a onstitutive manner and is not overtly sensitive to voltage. Similarly to all ENa/Deg hannels, ENa is trimeri, being a heterotrimer omprised of three similar subunits (a, b, and g) enoded by distint genes (2,7 9). ll ENa/Deg subunits share a ommon seondary and tertiary struture, i.e., a large extraellular domain separated from shorter ytosoli NH 2 - and OOH-tails by two transmembrane domains (TM1 and TM2). ENa serves an important physiologial funtion beause its ativity is limiting for Na þ transport aross epithelial barriers, partiularly in the kidney (4 6). onsequently, ENa is a ritial end-effetor of homeostati ontrol systems suh as the rennin-ngii-aldosterone system, whih governs blood pressure through feedbak regulation of systemi Na þ levels. The ritial importane of this hannel for proper ontrol of blood pressure is apparent when one onsiders that gain and loss of ENa funtion Submitted November 12, 21, and aepted for publiation February 28, 211. *orrespondene: stokand@uthssa.edu Editor: Indira M. Raman. result in inreases and dereases in blood pressure, respetively, assoiated with improper Na þ onservation and wasting by the kidney (1 13). Pseudohypoaldosteronism type 1 (PH-1) is an inherited renal Na þ wasting disorder that results from loss of ENa funtion (11 13). Every member of the ENa/Deg family ontains an absolutely onserved His-Gly sequene in its NH 2 -terminal ytosoli region just preeding TM1 (11,14 16). These residues are ritial for hannel funtion and their loss leads to dereased hannel ativity resulting from a derease in the hannel open probability (P o ) (11,1). point mutation resulting in substitution of the ritial Gly residue in human ENa (hena) auses PH-1 (11). However, beyond the fat that this dereases the P o, as tested at a single membrane potential, little is known about the mehanism that auses loss of funtion in ENa harboring this PH-1-ausing substitution. The reent solving of the rystal struture for the transmembrane and extraellular domains of hiken SI1 shed muh light on the struture of these portions of ENa/Deg hannels (8,9). In omparison, less is known about the struture and relative position of the ytosoli portions of ENa to inlude the neessary His-Gly funtional element that is ritial for normal P o and is disrupted in some forms of PH-1. Kir hannels ontain a girdle-like struture that fits into the intraellular mouth of their pore (17 19). This struture onsists of G-loops at the apex of the ytoplasmi domains and funtions as a flexible diffusion barrier, or gate, in the Ó 211 by the iophysial Soiety 6-349/11/4/193/1 $2. doi: 1.116/j.bpj

2 PH-1 Mutations ause Voltage Dependene 1931 K þ ondution pathway. Mutations that disrupt G-loops result in loss of funtion (18,19). Indeed, suh a mutation is ausative for ndersen-tawil syndrome, a form of inherited periodi paralysis and ventriular arrhythmias. The intraellular mouth of the pore of the M2 proton hannel is also ringed by a girdle-like struture (2 24). In this hannel, the indole rings in the side hains of Trp residues loated at the base of transmembrane domains form ation-p interations with the imidazolium rings in side hains of ritial His residues to stabilize the girdle and thus affet gating. The side hains of these ritial Trp residues in M2 proton hannels jut into the aqueous environment in the mouth of the pore. Of interest, ENa/Deg hannels ontain a onserved Trp at the intraellular base of TM1 (2). In the rystal struture of SI1, the side hains of these Trp residues jut into the aqueous mouth of the pore (9). onsidering the struture of Kir and M2 proton hannels, it is possible that the homologous Trp residues in ENa interat with ritial His residues to form a girdle-like struture that is important for gating. To explore this hypothesis, we studied the mehanism that underlies loss of funtion in PH-1-ausing substitutions in ENa. We found that as a result of PH-1-ausing substitutions, ENa beomes voltage-dependent and the P o is signifiantly dereased at physiologial potentials. It is the loss of a ritial funtional element upon substitution rather than the introdution of novel struture that results in this phenotype. Moreover, the voltage sensitivity that results from mutation of the onserved His-Gly residues is akin to that observed upon substitution of the onserved Trp at the base of TM1. These data are onsistent with the notion that ENa also has a girdle-like struture just below the intraellular mouth of its pore that plays an important role in hannel gating. MTERILS ND METHODS In this study we used standard reagents, praties, and eletrophysiology methods to investigate wild-type and mutant ENa (2 27). omplete desription of the methods used is provided in the Supporting Material. hena; α, β, γ 6 mv 4 mv - mv ms hena; αg7s, β, γ hena; α, βg37s, γ amil 2 p 2 mse amil amil RESULTS PH-1-ausing mutations in hena derease hannel ativity Fig. 1 shows representative marosopi Na þ urrents before and after treatment with 1 mm amiloride (amil., indiated by arrow) for HO ells expressing wild-type hena (top, a þ b þ g) and hena ontaining PH- 1-ausing missense substitutions, suh as G7S (middle) and G37S (bottom) in the a-hena and b-hena subunits, respetively. urrents were evoked with a voltage ramp from 6 to mv from a holding potential of 4 mv, whih is near E Na for the physiologial pipette and bath solutions used in these experiments. miloride is a broadspetrum, open-hannel inhibitor for most hannels, partiularly ENa, in the ENa/Deg family (4,). s shown in Fig. 1, the introdution of a PH-1-ausing mutation into at least one hena subunit resulted in a signifiant derease in hannel ativity, as expeted. This finding is onsistent with previous studies showing that PH- 1-ausing substitutions (e.g., G37S in b-hena) in hena result in a loss of hannel funtion (11,12). In addition, it is onsistent with previous investigations of the onserved His-Gly sequene in whih substitution of these residues dereased hannel ativity (11,16). hena ontaining PH-1-ausing mutations senses voltage as a result of voltage-dependent hanges in P o In previous studies (11,1), the derease in ativity for hena ontaining PH-1-ausing mutations was investigated at steady state, at a single holding potential. Thus, little was revealed about the mehanism that auses loss of funtion, and how these mutant hannels behave over a range of voltages. To address these issues, we generated a whole-ell marosopi urrent-voltage (I/V) relation for hena harboring a PH-1-ausing substitution, ag7s, and ompared it with that of wild-type hena. Fig. 2 shows representative families of marosopi Na þ urrents in symmetrial Nal solutions, evoked by 2 mv voltage steps up to mv and down to 2 mv from a holding urrent density (p/pf) 2 1 * * hena αg7s βg37s FIGURE 1 PH-1-ausing mutations in hena derease hannel ativity. () Representative marosopi Na þ urrents from HO ells expressing wild-type hena a-, b-, and g-subunits (top) or subunits engineered to ontain PH-1-ausing mutations (middle and bottom) oexpressed with the omplementary wild-type subunits before and after addition of 1 mm amiloride (arrow). Whole-ell, marosopi urrents evoked with a voltage ramp (shown in inset) from 6 mv to mv from a holding potential of 4 mv. For these experiments, the bath and pipette [Na þ ] were asymmetrial at 1 and mm, respetively. () Summary graph of (amiloride-sensitive) urrent density at 8 mv (from voltage ramps) for voltage-lamped HO ells, similar to that in panel, expressing wild-type hena and hena harboring PH-1-ausing mutations in either the a- or b-subunit. The number of experiments for eah group is indiated. urrent was measured with voltage ramps. * Signifiant (P <.) derease versus wild-type hena. iophysial Journal (8)

3 1932 Kuher et al. FIGURE 2 hena harboring the PH-1-ausing hena, mutation, G7S, senses voltage. () Families of marosopi Na þ urrents from representative -2-1 HO ells expressing wild-type (top) and hena harboring the ag7s PH-1 mutation (bottom). - hena αg7s at 1 urrents were evoked by progressive 2 mv voltage αg7s at 2 steps from a holding potential of mvup to mv αg7s,β, γ I / I and down to 2 mv (voltage protool shown in αg7s -1 inset). For these experiments, the bath and pipette [Na þ ] were symmetrial at 1 mm. () Marosopi p mv 8 mv I/V relations for HO ells expressing 2 ms mv wild-type (blak line) and hena with the ag7s mutation (gray line). The I/V relations were generated -2 mv from experiments similar to that in panel. For presentation, the urrent was normalized to urrent at mv. () Steady-state G-V relations fitted with the oltzmann equation for HO ells expressing wild-type (open squares) and hena ontaining ag7s (solid squares: developed from steady-state urrent at time 1 using voltage steps; open irles: developed from instantaneous tail urrents at 8 mvat time 2). For presentation, ondutane was normalized to the maximum of the fit. For and, n R 9 for eah group. G/G max potential of mv for HO ells expressing wild-type (top, a þ b þ g) and mutant hena ontaining an a-subunit with the G7S substitution oexpressed with wild-type b- and g- subunits (bottom). Fig. 2 shows the resulting I/V relations for these hannels at steady state. s expeted, and onsistent with previous findings (4,2), wild-type hena has a linear I/V relation in symmetrial Nal solutions. Surprisingly, the I/V for hena ontaining the PH-1-ausing substitution has a notieable voltage dependene. The voltage dependene of steady-state urrents (at time 1) and tail urrents (at time 2, at 8 mv) for wild-type (open boxes) and mutant hena ontaining the ag7s substitution (solid boxes and open irles) is quantified in Fig. 2. learly, hena does not respond to voltage. In ontrast, hena harboring a PH-1-ausing mutation has pronouned voltage dependene, with ativity inreasing signifiantly at hyperpolarizing potentials. We found no differene when we quantified the voltage dependene with voltage steps versus tail urrents. The half-ativation potential for hena ontaining ag7s is estimated to be mv. s shown in Fig. S1, the time onstants of ativation and deativation for ag7s hannels are and ms, respetively, at mv. Voltage dependene at a marosopi level an arise from differenes in permeability, seletivity, single-hannel ondutane, or an effet of voltage on hannel gating leading to hanges in P o. To distinguish among these possibilities, we next studied wild-type and hena ontaining the ag7s substitution at the single-hannel level in exised, outside-out pathes. Representative, ontinuous urrent traes for hena (top) and hena ontaining the ag7s substitution (bottom) at voltages spanning 8 to 8 mv are shown in Fig. 3. Traes were obtained from pathes pulled from HO ells exposed to symmetrial Lil solutions. s is learly demonstrated by the I/V relations shown in Fig. 3, wild-type and mutant hena have similar single-hannel ondutane ( and ps, respetively) and, to the extent tested, seletivity and permeability values. The ause of the voltage dependene is revealed by the P o -V urves shown in Fig. 3 : the P o of wild-type hena does not hange over the range of voltages probed here, whereas that for hena ontaining ag7s inreases as a funtion of hyperpolarizing potentials. The potential where P o ¼. for this mutant is estimated to be 14 4 mv. These singlehannel results support the idea that voltage-sensitive hanges in P o are the primary mehanism underlying the voltage sensitivity and lower resting ativity at physiologial potentials for hena that harbor this PH-1 substitution. The voltage sensitivity in ENa aused by PH-1 substitutions of the ritial His-Gly sequene is speies- and subunit-independent fter establishing the mehanism underlying loss of funtion for hena ontaining the ag7s substitution, we wondered whether the properties introdued by this substitution were unique to ENa from a speifi speies or to a partiular hannel subunit. To investigate this issue, we introdued homologous PH-1-ausing mutations into individual mouse ENa (mena) subunits and expressed them with omplementary wild-type mena subunits. Fig. 4 shows representative marosopi Na þ urrents () and the resulting I/V relations () from HO ells expressing mena ontaining ag9s (top), bg37s (middle), or gg4s (bottom). Symmetrial Nal solutions were used in these experiments. Fig. 4 ompares the steady-state ativity (at 8 mv) of mutant mena ontaining homologous PH-1-ausing mutations in a single subunit with that of wild-type mena. Similarly to hena, mena ontaining a PH-1 substitution has dereased ativity. s is lear from the ondutane-voltage (G-V) urves shown in Fig. 4 D (whih were developed from the respetive marosopi I/V relations in Fig. 4 ), the underlying ause iophysial Journal (8)

4 PH-1 Mutations ause Voltage Dependene 1933 hena; α, β, γ hena; αg7s, β, γ 8 mv 6 mv 4 mv -4 mv -6 mv -8 mv. p 2 se 8 mv 6 mv 4 mv -4 mv -6 mv i, p P o hena αg7s hena αg7s FIGURE 3 hena ontaining ag7s gates in a voltage-dependent manner with inreased P o at hyperpolarizing potentials. () Families of representative single-hannel urrent traes for wildtype (top) and mutant (bottom) hena in outside-out pathes stepped from 8 mv to 8 mv. The bath and pipette [Li þ ] were symmetrial for these experiments. Inward Na þ urrent is downward;, losed state. () Single-hannel I/V relations for wild-type (blak squares) and ag7s (gray irles) hena in outside-out pathes. Data were obtained from experiments idential to that in panel ; n R 3 for eah group. () Plot showing the ENa P o as a funtion of voltage for wild-type (blak squares) and mutant hannels ontaining the ag7s mutation (gray irles). Data were fit by the oltzmann equation. Data were obtained from experiments idential to that in panel ; n R 3 for eah group. -8 mv of this derease in ativity, at least for mena ontaining ag9s (estimated V 1/2 ¼ mv) and gg4s (estimated V 1/2 ¼ 18 11), is the voltage sensitivity introdued by substitution. This voltage sensitivity is akin to that seen in hena ontaining ag7s. It is less ertain that this also is the underlying ause of dereased ativity in mena ontaining bg37s, sine our investigation of the mehanism for this mutant was onstrained by experimental limitations (we were unable to probe potentials more hyperpolarized than 2 mv). However, voltage dependene and lear ativation kinetis begin to emerge at potentials near 2 mv for this mutant. It is possible that the ativity of mena ontaining bg37s would inrease upon further hyperpolarization. Nevertheless, these results are most onsistent with the idea that the effets of substituting the homologous Gly residues in ENa are responsible, in a speies- and subunit-independent manner, for the PH-1 phenotype leading to voltage sensitivity, resulting in less ativity at physiologial potentials. Loss of the ritial Gly residue rather than introdution of novel side hains results in the PH-1 phenotype s shown in Fig., a His-Gly sequene is ommon to the NH 2 -terminal ytoplasmi region just preeding TM1 in every ENa/Deg subunit exept human SI2b, whih ontains an rg in plae of His. This onservation led us to ask whether the loss-of-funtion phenotype in hannels harboring subunits with the PH-1-ausing substitution of the ritial Gly arises from loss of this residue or from the introdution of a speifi type of side hain when the residue is replaed. To investigate this issue, we assayed the ativity and voltage dependene of mena ontaining a-subunits in whih G9 was substituted with distint amino aids bearing widely different side hains. Fig. 6, and, show representative marosopi urrents and the assoiated I/V relations for HO ells expressing mena that ontain a-subunits with G9 substituted with different amino aids (alanine and ysteine). Studies were performed with symmetrial Nal. Inward retifiation is apparent in both mutants. Fig. 6 ompares the steady-state ativity (at 8 mv) of mena ontaining differential substitution of ag9 with S,,, and W ompared with wild-type mena. Every mutant had signifiantly less ativity than the wild-type, and ag9w was nonfuntional (as far as tested). These findings are onsistent with the notion that loss of the ritial Gly residue, rather than the introdution of a novel residue with a speifi type of side hain, results in this phenotype. Thus, Gly at this position is an absolute requirement for normal hannel ativity. Moreover, as indiated by the G-V urves in Fig. 6 D, substitution of G9 in a-mena always resulted in a similar voltage dependene when a funtional hannel was produed (estimated V 1/2 for G9S, G9, and G9 ¼ mv, , and 23 mv, respetively). This voltage dependene underlies the notable inward retifiation for these mutants that is apparent in Fig. 6. In addition, the iophysial Journal (8)

5 1934 Kuher et al. mena; αg9s, β, γ 2 mena; α, βg37s, γ αg9s I / I - urrent density (p/pf) mena * * * αg9s βg37s γg4s 2 p 2 ms mena; α, β, γ G4S βg37s I / I - D 1 G/G max -2-2 αg9s γg4s. -4 γg4s -6 I / I FIGURE 4 PH-1 mutation results in a onsistent ENa phenotype aross speies and hannel subunits. () Families of marosopi Na þ urrents from representative HO ells expressing mena harboring PH-1-ausing mutations in the a (top; G9S), b (middle; G37S), and g (bottom; G4S) subunits. urrent was evoked by progressive 2 mv voltage steps from a holding potential of mv up to mv and down to 2 mv. For these experiments, the bath and pipette [Na þ ] were symmetrial at 1 mm. () Marosopi I/V relations for HO ells expressing wild-type (blak lines) and mutant mena (gray lines) ontaining ag9s (top), bg37s (middle), or gg4s (bottom). The I/V relations were generated from experiments similar to that in panel. For presentation, urrent was normalized to urrent at mv. () Summary graph of (amiloride-sensitive) urrent density at steady state at 8 mv for HO ells, similar to that in panel, expressing wild-type mena and mena harboring PH-1-ausing mutations in the a-, b-, or g-subunit. The number of experiments for eah group is indiated. * Signifiant (P <.) derease versus wild-type mena. (D) Steady-state G-V relations were fitted with the oltzmann equation for HO ells expressing wild-type (open boxes) and mena ontaining ag9s (blak squares) and gg4s (open irles). Data were obtained from experiments idential to that in panel. For presentation, ondutane was normalized to the maximum of the fit. For and D, n R 6 for eah group. dependene on hyperpolarizing voltages for signifiant ativity in these mutant hannels is onsistent with voltage dependene leading to low ativity at physiologial potentials. The region that inludes the onserved His-Gly sequene is important for normal hannel funtion We next sanned through the region that inluded the onserved His-Gly sequene to determine whether nearby residues/positions are also ritial for normal ENa funtion. Fig. 7 shows representative marosopi urrents () and the orresponding I/V relations () for mena ontaining a-subunits with T92 (left), I93 (left middle), H94 (right middle), or 96 (right) substitutions. s shown in Fig., neither the Thr at position 92, the Ile at 93, nor the la at 96 is onserved in ENa/Deg paralogs, although these residues are onserved within the individual subunit orthologs. s expeted, substitution of H94 resulted in inward retifiation. Substitution of the Thr and the la three positions upstream and one downstream, respetively, of the ritial G9 in a-mena had no effet on I/V relations. Substitution of I93, though, also resulted in inward retifiation. The steady-state ativities for mutant hannels at 8 mv are summarized in Fig. 7. In ontrast to the onserved His-Gly sequene, although substitution of I93 resulted in inward retifiation, it did not have a major effet on steady-state ativity. Fig. 7 D reports the G-V relations for these mutants. s for G9, signifiant voltage dependene iophysial Journal (8)

6 PH-1 Mutations ause Voltage Dependene TIHG α-mena NTHGP β-mena NTHG γ -mena RLHGL fsi2 KLRGL hsi2b SMHGL hsi3 SHGI ME-4 KFHGI ssi1a NLHGV ssi1b TLHGM zsi1.3 FIGURE onsensus sequene around the onserved HG motif in the ytosoli amino-terminus of ENa/Deg subunits. The sequenes shown for a-, b-, and g-ena subunits are absolutely onserved among ENa orthologs but not ENa/Deg paralogs. Examples of paralogs that ontain the different amino aids at 3, 2, and þ1 reported to date are shown below. Numbering for a-mena. is the underlying ause of the inward retifiation of H94 (estimated V 1/2 ¼ mv) and I93 mutants (estimated V 1/2 ¼ mv). lso inluded in Fig. 7 D is the G-V urve for mena bearing the W112 mutation in the a-subunit. s reported previously (2), this mutation, similarly to PH-1-ausing substitutions of G9, leads to voltage dependene and dereased ativity. Of interest, the ativation time upon hyperpolarization for the I93 substitution (Fig. 7 E) is>1 times slower than that for either the H94 or G9 mutation. t 6 mv, t at for I93, H94, and G9 ¼ 3.4.3,.2.2, and.2.3 s, respetively. Fig. 8 shows representative, ontinuous single-hannel urrent traes for ai93 () and ah94 () mutants in outside-out pathes in symmetrial Lil. s is lear in these representative traes and the summary P o -V plot in Fig. 8, voltage-dependent hanges in P o underlie voltage sensitivity at the marosopi level. The steady-state P o at 8 mv for I93 mutants approahes that of the wild-type at this voltage, whih explains the normal marosopi ativity. The relatively slow time of ativation for mena ontaining an ai93 substitution allows definitive determination of the mehanism underlying voltage sensitivity Fig. 9 shows four representative single-hannel urrent traes from outside-out pathes pulled from HO ells expressing mena with the ai93 mutation. Experiments were performed in symmetrial Lil. Pathes were rapidly transitioned from 6 mv to 6 mv and bak again, with instantaneous urrent aptured diretly after the voltage step. These representative experiments and the summary graph of instantaneous NP o (hannel ativity where N is the number of funtional hannels in the pathed membrane and P o is the mean open probability these hannels have) as a funtion of time diretly after the step in voltage to 6 mv (Fig. 9 ) learly demonstrate that hanges in singlehannel P o our immediately. The time onstants of ativation and deativation for I93 exposed to this voltage protool are and s, respetively. These time onstants agree well with the marosopi urrent results for this mutant presented in Fig. 7 E. Moreover, these results provide inontrovertible evidene of mehanism. FIGURE 6 Loss of a ritial Gly residue in the mena; αg9, β, γ 3 ytosoli portion of mena results in the PH-1 phenotype. () Families of marosopi Na þ urrents from representative voltage-lamped 2 - HO ells expressing mena ontaining ag9 substituted with la (top) or ys (bottom). -1 I / I - urrents were evoked by progressive 2 mv * * * * voltage steps from a holding potential of mv -1 3 p αg up to mv and down to 2 mv. For these 2 ms -2 experiments, the bath and pipette [Na þ ] were symmetrial at 1 mm. () Marosopi I/V relations for HO ells expressing wild-type (blak D mena; αg9, β, γ 1. lines) and mutant (gray lines) mena ontaining G/G max ag9 (top) orag9 (bottom). The I/V relations were generated from experiments similar to that - αg9s. shown in Fig.. For presentation, the urrent αg9-1 I / I - αg9 was normalized to urrent at mv. () Summary graph of (amiloride-sensitive) urrent -1 αg9 density at steady state at 8 mv for voltagelamped HO ells (as in Fig. ) expressing wild-type mena and mena ontaining ag9s, ag9, ag9, or ag9w. The number of experiments for eah group is indiated. * Signifiant (P <.) derease versus wild-type mena. (D) Steady-state G-V relations were fitted with the oltzmann equation for HO ells expressing mena ontaining ag9s (open squares), ag9 (gray irles), or ag9 (blak squares). Data are from experiments idential to that in panel. For and D, n R 6 for eah group. urrent density (p/pf) mena αg9s αg9 αg9 αg9w iophysial Journal (8)

7 1936 Kuher et al. αt92 (-3) αi93 (-2) αh94 (-1) α96 (+1) 1 n 2 ms 1 n 2 s n 2 ms - 1 n 2 ms - - urrent density (p/pf) 3 2 αt92 mena I / I - * * αt92 αi93 αh94 αg9 α96 αi93 D αi93 αh94 αg9 α96 αw112 I / I -. αh G/G max -4-2 E I / I- I / I max -1-1 α96 I93 I93 G9-2 I / I - τ at, se 2 4 αi93 * αh94 αg9 H94 1 time,se 1 or 1 FIGURE 7 Disruption of the Ile (ai93) and His (ah94) residues immediately upstream of the ritial PH-1 Gly (ag9) also leads to voltage dependene reminisent of mutation of the onserved Trp (aw112) residue at the intraellular base of TM1. () Families of marosopi Na þ urrents from HO ells expressing mena ontaining a-subunits with T92 (left), I93 (left middle), H94 (right middle), or 96 (right) substitution. The relative position to the ritial Gly (G9) is indiated. urrents evoked by progressive 2 mv voltage steps from a holding potential of mv up to mv and down to 2 mv. For these experiments, the bath and pipette [Na þ ] were symmetrial at 1 mm. () Marosopi I/V relations for HO ells expressing wild-type (blak lines) and mutant (gray lines) mena ontaining a-subunits with T92 (left), I93 (left middle), H94 (right middle), or 96 (right) substitution. The I/V relations were generated from experiments similar to that in panel. For presentation, urrent was normalized to urrent at mv. () Summary graph of (amiloride-sensitive) urrent density at steady state at 8 mv for voltage-lamped HO ells (as in Fig. 6 ) expressing wild-type mena and mena ontaining a-subunits with T92, I93, H94, G9, or 96 substitution. * Signifiant (P <.) derease versus wild-type mena. (D) Steady-state G-V relations were fitted with the oltzmann equation for HO ells expressing mena ontaining a-subunits with I93 (gray triangle), H94 (open irle), G9 (blak box), 96 (open box), or W112 (dashed line) substitution. Data are from experiments idential to that shown in panel.(e) Typial ativation kinetis at 2 mv for normalized (to maximums at steady state) marosopi Na þ urrents arried by mena ontaining a-subunits with I93, H94, or G9 substitution. urrent for I93 is shown at both 1 s (blak line) and 1 s (gray line; noted with arrow) timesales. Inset shows mean t ativation at 6 mv for the respetive mutants. For D and inset in E, n R 6 for eah group. DISUSSION The results of this study onfirm that substituting a onserved Gly residue in the ytoplasmi pre-tm1 region of the NH 2 -terminus of hena subunits auses loss of funtion (11,14 16). We further show that this effet is not subunit-dependent, sine dereases in ativity an arise from mutation of any one of the three omponent subunits of the hannel. We interpret this as indiating that the onserved Gly residue within eah subunit serves a ommon funtion. Moreover, loss of funtion arises from homologous mutations in mena subunits, suggesting that the effet is ommon to all ENa/Deg hannel proteins independently of speies. These findings demonstrate the ritial importane of this Gly residue for ativity, and are onsistent with the idea that it is a key funtional element within ENa/Deg hannels. Having a Gly residue at this key position is ritial for hannel funtion beause replaing this residue with different amino aids with distint side hains leads to similar dereases in hannel ativity. Replaing the small, nonpolar Gly residue at this position with a similarly small, nonpolar la residue is equivalent to inserting bulkier, hydrophobi ysteine and tryptophan residues. Thus, the differene between having a Gly or la at this position is enough to ause loss of funtion, suggesting a rigid struture-funtion requirement at this site. surprising finding of our study is that missense substitution of the onserved Gly residue results in inward retifiation of marosopi urrents due to notable voltage dependene, suh that the ativity of mutant hannels is low at iophysial Journal (8)

8 PH-1 Mutations ause Voltage Dependene mv +6 mv +4 mv -4 mv -6 mv mena; α I93, β, γ mena; αh94, β, γ.6 W112 P o.4.2 I93 H94-8 mv. p 2 se FIGURE 8 mena ontaining a-subunits with I93 and H94 substitutions gate in a voltage-dependent manner. Families of representative singlehannel urrent traes from HO ells for reombinant mena ontaining ai93 () and ah94 () in outside-out pathes stepped from 8 mv to 8 mv. The bath and pipette [Li þ ] were symmetrial for these experiments. Inward Na þ urrent is downward;, losed state. () Plot showing ENa P o as a funtion of voltage for wild-type (blak squares) and mutant mena ontaining aw112 (open irles), ai93 (open squares), or ah94 (blak irles). Data were fit by the oltzmann equation and obtained from experiments idential to that shown in panels and ; n R 4 for eah group. physiologial potentials but inreases with hyperpolarization. The ause of this voltage sensitivity is voltage-dependent gating rather than differenes in single-hannel ondutane or seletivity with P o inreasing as a funtion of membrane hyperpolarization. The onserved Gly mutated in some forms of PH-1 is preeded in the primary sequene by a onserved His residue. In similarity to the ritial role played by Gly, missense substitution of this onserved His results in voltage-dependent gating and dereases in hannel ativity. This finding is onsistent with the previous observation that marosopi ativity is dereased for hannels that are mutant in this His (1,16). Substitution of the Ile in a-mena two positions upstream of the ritial Gly residue slows the ativation and deativation kinetis of voltage-dependent hanges in P o enough to allow quantitation of hanges in instantaneous P o immediately after hanges in potential. This is important beause it definitively establishes the mehanism that underlies loss of funtion: a voltage-dependent derease in P o. The derease in ativity and underlying mehanism of voltagedependent gating for ENa ontaining substitution of the key His-Gly residues is akin to that observed for ENa ontaining a missense substitution of a onserved Trp residue at the base of TM1 (2), suggesting the possibility of funtional onvergene and/or interation between these residues/regions of the hannel. The mehanism underlying voltage dependene in the latter mutant form of the hannel also inludes voltage-dependent hanges in P o with hyperpolarizing voltages relieving blok of the pore by intraellular Na þ. We are not the first to report that missense substitutions of onserved His and Gly residues in the ytoplasmi, pre- TM1 region of ENa/Deg subunits ause loss of funtion. Indeed, this has been established (11,12,14,1). Of importane, though, we expand on this understanding by delving into the underlying mehanism that auses dereases in hannel ativity, and disuss the assoiated struture-funtion impliations in the ontext of what is known about the struture of ENa and other ion hannels. s noted in the Introdution, Kir and M2 proton hannels ontain a girdle-like struture that runs around the intraellular mouth of their pores (17 19,21 24). These strutures play a role in gating and involve the interations of ytoplasmi regions of the hannels with residues in transmembrane domains. The results we obtained here and in a previous work onerning an interfaial Trp residue at the base of TM1 (2) suggest that ENa may have a similar struture. This interpretation is just one of many possibilities, but to us it seems the most likely and well supported beause it is onsistent with all urrent findings. There are several ommonalities in how ENa funtion hanges upon substitution of the residues in the onserved His-Gly sequene and the onserved Trp at the base of TM1. For instane, both lead to a derease in hannel ativity as a onsequene of the emergene of voltage-dependent gating, with P o being low at physiologial potentials and inreasing with hyperpolarization. It is also likely that both share a ommon mehanism of ativation involving relief of pore blok upon hyperpolarization. onsidering the struture-funtion relations in M2 proton hannels, whih also have similar onserved His and Trp residues that interat to form the girdle-like struture inside the mouth of this hannel (whih is important for gating) (21 24), a logial suggestion is that these residues in ENa also interat in a manner reminisent of interations in M2 proton hannels to influene gating. Suh an interation in ENa/Deg proteins may struturally require a small, nonpolar residue, namely Gly, to immediately follow the onserved His to allow normal hannel funtion and gating. This senario is onsistent with the reent finding that the side hains of the interfaial Trp residues at the base of iophysial Journal (8)

9 1938 Kuher et al. α I mv - 6 mv SUPPORTING MTERIL Methods, one table, and one figure are available at biophysj/supplemental/s6-349(11) p 2 se This researh was supported by the National Institutes of Health (grant R1DK771) and the merian Heart ssoiation (Established Investigator ward 644N to J.D.S.). Instantaneous NP o seonds FIGURE 9 The relatively slow times of ativation and deativation for mena ontaining ai93 substitution enables doumentation at the single-hannel level of voltage-dependent gating for a substitution similar to PH-1-ausing mutations. () Four representative single-hannel urrent traes in outside-out pathes held at 6 (left) and 6 mv (right) for mutant mena harboring the ai93 substitution. The displayed urrents immediately follow hanges in holding potential. The bath and pipette [Li þ ] were symmetrial for these experiments. Inward Na þ urrent is downward;, losed state. () Dairy plot showing hanges in instantaneous (2 s bins), mean NP o for ai93 hannels as they deativate immediately after a voltage step from 6 to 6 mv. Data were obtained from n R 4 experiments idential to those shown in panel. TM1 protrude into the aqueous environment of the intraellular mouth of the pore, where they are available to interat with other residues (9). In the ontext of suh a disussion, the novel (to our knowledge) findings presented here are the first to suggest that the ytoplasmi, intraellular NH 2 -terminal regions of ENa/Deg hannel proteins may have a ritial struture that is apable of impating hannel gating. One possibility is that the funtional elements within this region of ENa may be part of a struture reminisent of the girdle-like struture that rings the intraellular mouths of Kir and M2 proton hannels. The disruption of suh a struture would then interrupt normal hannel gating, leading to the loss of funtion that is apparent in PH-1 mutants. REFERENES 1. anessa,. M., J. D. Horisberger, and.. Rossier Epithelial sodium hannel related to proteins involved in neurodegeneration. Nature. 361: anessa,. M., L. Shild,.,.. Rossier miloride-sensitive epithelial Na þ hannel is made of three homologous subunits. Nature. 367: Waldmann, R., G. hampigny,., M. Lazdunski Moleular loning and funtional expression of a novel amiloride-sensitive Na þ hannel. J. iol. hem. 27: Kellenberger, S., and L. Shild. 22. Epithelial sodium hannel/degenerin family of ion hannels: a variety of funtions for a shared struture. Physiol. Rev. 82: Garty, H., and L. G. Palmer Epithelial sodium hannels: funtion, struture, and regulation. Physiol. Rev. 77: enos, D. J., and.. Stanton Funtional domains within the degenerin/epithelial sodium hannel (Deg/ENa) superfamily of ion hannels. J. Physiol. 2: Starushenko,., E. dams,., J. D. Stokand. 2. Epithelial Na þ hannel subunit stoihiometry. iophys. J. 88: Jasti, J., H. Furukawa,., E. Gouaux. 27. Struture of aid-sensing ion hannel 1 at 1.9 resolution and low ph. Nature. 449: Gonzales, E.., T. Kawate, and E. Gouaux. 29. Pore arhiteture and ion sites in aid-sensing ion hannels and P2X reeptors. Nature. 46: Lifton, R. P Geneti determinants of human hypertension. Pro. Natl. ad. Si. US. 92: Gründer, S., D. Firsov,.,.. Rossier mutation ausing pseudohypoaldosteronism type 1 identifies a onserved glyine that is involved in the gating of the epithelial sodium hannel. EMO J. 16: hang, S. S., S. Grunder,., R. P. Lifton Mutations in subunits of the epithelial sodium hannel ause salt wasting with hyperkalaemi aidosis, pseudohypoaldosteronism type 1. Nat. Genet. 12: Hummler, E., P. arker,.,.. Rossier mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism. Pro. Natl. ad. Si. US. 94: Tavernarakis, N., J. K. Everett,., M. Drisoll. 21. Strutural and funtional features of the intraellular amino terminus of DEG/ENa ion hannels. urr. iol. 11:R2 R Gründer, S., N. F. Jaeger,.,.. Rossier Identifiation of a highly onserved sequene at the N-terminus of the epithelial Na þ hannel a subunit involved in gating. Pflugers rh. 438: Hong, K., I. Mano, and M. Drisoll. 2. In vivo struture-funtion analyses of aenorhabditis elegans ME-4, a andidate mehanosensory ion hannel subunit. J. Neurosi. 2: Nishida, M., M. adene,., R. MaKinnon. 27. rystal struture of a Kir3.1-prokaryoti Kir hannel himera. EMO J. 26: Pegan, S.,. rrabit,., S. hoe. 2. ytoplasmi domain strutures of Kir2.1 and Kir3.1 show sites for modulating gating and retifiation. Nat. Neurosi. 8: Ma, D., X. D. Tang,., P.. Welling. 27. n ndersen-tawil syndrome mutation in Kir2.1 (V32M) alters the G-loop ytoplasmi K þ ondution pathway. J. iol. hem. 282: iophysial Journal (8)

10 PH-1 Mutations ause Voltage Dependene Witter, R., F. Nozirov,., R. Fu. 28. Solid-state 19F NMR spetrosopy reveals that Trp41 partiipates in the gating mehanism of the M2 proton hannel of influenza virus. J. m. hem. So. 13: Shnell, J. R., and J. J. hou. 28. Struture and mehanism of the M2 proton hannel of influenza virus. Nature. 41: Takeuhi, H.,. Okada, and T. Miura. 23. Roles of the histidine and tryptophan side hains in the M2 proton hannel from influenza virus. FES Lett. 2: Stouffer,. L., R. harya,., W. F. DeGrado. 28. Strutural basis for the funtion and inhibition of an influenza virus proton hannel. Nature. 41: Tang, Y., F. Zaitseva,., L. H. Pinto. 22. The gate of the influenza virus M2 proton hannel is formed by a single tryptophan residue. J. iol. hem. 277: Pohynyuk, O., V. Kuher,., J. Stokand. 29. Intrinsi voltage dependene of the epithelial Na þ hannel is masked by a onserved transmembrane domain tryptophan. J. iol. hem. 284: Pohynyuk, O., Q. Tong,., J. D. Stokand. 27. Moleular determinants of PI(4,)P2 and PI(3,4,)P3 regulation of the epithelial Na þ hannel. J. Gen. Physiol. 13: Starushenko,., R. E. ooth,., Q. Tong. 26. Funtional reonstitution of the human epithelial Na þ hannel in a mammalian expression system. Methods Mol. iol. 337:3 13. iophysial Journal (8)