Mutations in the pore region modify epithelial sodium channel gating by shear stress

Previous studies have shown that epithelial Na+ channels (ENaCs) are activated by laminar shear stress (LSS). ENaCs with a high intrinsic open probability because of a mutation (betaS518K) or covalent modification of an introduced Cys residue (alphaS580C) in the pre-second transmembrane domain (pre-M2) were not activated by LSS, suggesting that the pre-M2 region participates in conformational rearrangements during channel activation. We examined the role of the pore region of the alpha-subunit in channel gating by studying the kinetics of activation by LSS of wild-type ENaC and channels with Cys mutations in the tract Ser576-Ser592. Whole cell Na+ currents were monitored in oocytes expressing wild-type or mutant ENaCs prior to and following application of LSS. Following a 2.2-s delay, a monoexponential increase in Na+ currents was observed with a time constant (tau) of 8.1 s in oocytes expressing wild-type ENaC. Cys substitutions within the alpha-subunit in the tract Ser580-Ser589 resulted in: (i) a reduction (Ser580-Trp585, Gly587) or increase (Ser589) in delay times preceding channel activation by LSS, (ii) an increase (Gln581, Leu584, Trp585, Phe586, Ser588) or decrease (Ser589) in the rate of channel activation, or (iii) a decrease in the magnitude of the response (Ser583, Gly587, Leu584). Cys substitutions at a putative amiloride-binding site (alphaSer583 or betaGly525) or within the selectivity filter (alphaGly587) resulted in a reduction in the LSS response, and exhibited a multiexponential time course of activation. The corresponding gamma-subunit mutant (alphabetagammaG542C) had a minimal response to LSS and exhibited a high intrinsic open probability. These data suggest that residues in the pore region participate in the sensing and/or transduction of the mechanical stimulus that results in channel activation and are consistent with the hypothesis that the ENaC pore region has a key role in modulating channel gating.     

Second transmembrane domains of ENaC subunits contribute to ion permeation and selectivity

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.     

Side chain orientation of residues lining the selectivity filter of epithelial Na+ channels

Epithelial Na(+) channels (ENaCs) selectively conduct Na(+) and Li(+) but exclude K(+). A three-residue tract ((G/S)XS) present within all three subunits has been identified as a key structure forming a putative selectivity filter. We investigated the side chain orientation of residues within this tract by analyzing accessibility of the introduced sulfhydryl groups to thiophilic Cd(2+). Xenopus oocytes were used to express wild-type or mutant mouse alphabetagammaENaCs. The blocking effect of external Cd(2+) was examined by comparing amiloride-sensitive Na(+) currents measured by two-electrode voltage clamp in the absence and presence of Cd(2+) in the bath solution. The currents in mutant channels containing a single Cys substitution at the first or third position within the (G/S)XS tract (alphaG587C, alphaS589C, betaG529C, betaS531C, gammaS546C, and gammaS548C) were blocked by Cd(2+) with varying inhibitory constants (0.06-13 mm), whereas the currents in control channels were largely insensitive to Cd(2+) at concentrations up to 10 mm. The Cd(2+) blocking effects were fast, with time constants in the range of seconds, and were only partially reversible. The blocked currents were restored by 10 mm dithiothreitol. Mutant channels containing alanine or serine substitutions at these sites within the alpha subunit were only poorly and reversibly blocked by 10 mm Cd(2+). These results indicate that the introduced sulfhydryl groups face the conduction pore and suggest that serine hydroxyl groups within the selectivity filter in wild-type ENaCs face the conduction pore and may contribute to cation selectivity by participating in coordination of permeating cations.     

Epithelial sodium channel pore region. structure and role in gating

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.     

Characterization of the selectivity filter of the epithelial sodium channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed alpha, beta, and gamma. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-Val(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser(592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alphaG587C or alphaS589C were permeable to K(+), and mutation of a GSS tract (positions alpha587-alpha589) to GYG resulted in a moderately K(+)-selective channel. Our results suggest that the C-terminal portion of the pore region within the alpha-subunit contributes to the selectivity filter of ENaC.     

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.     

A molecular model of the inner pore of the Ca channel in its open state

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel¹ that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of ≠-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.     

Localization of the pH gate in Kir1.1 channels

We used cysteine-modifying reagents to localize the pH-sensitive gate in the renal inward-rectifier K(+) channel Kir1.1a (ROMK1). Cytoplasmic-side methanethiosulfonate (MTS) reagents blocked K(+) permeation in native Kir1.1 channels, expressed in Xenopus oocytes. Replacement of three cysteines in the N-terminus, C-terminus, and transmembrane domains eliminated this sensitivity to MTS reagents, as measured with inside-out macropatches. Reintroduction of one cysteine at 175-Kir1.1a in the second transmembrane domain allowed blockade of the open channel by the MTS reagents MTSEA, MTSET, and MTSES and by Ag(+). However, closure of the channel by low pH protected it from modification. Cysteine was also introduced into position G223, which is thought to line the cytoplasmic pore of the channel. MTSET blocked G223C in both the open and closed state. In contrast, MTSEA reduced G223C single-channel conductance from 40 to 23 pS but did not produce complete block. We conclude that cytoplasmic acidification induces a conformational change in the channel protein that prevents access of cysteine-modifying reagents, and presumably also K(+) ions, to the transmembrane pore from the cytoplasm. This is consistent with localization of the Kir1.1 pH gate at the helix bundle crossing near the cytoplasmic end of the transmembrane pore.     

A molecular model of the inner pore of the Ca channel in its open state

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel¹ that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of π-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.Key words: calcium channels, homology modeling, π-bulges, restrained minimization     

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.     

Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines.

In voltage- and cyclic nucleotide-gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the alpha subunit of cyclic nucleotide-gated channels from bovine rod, the pore loop is formed by the residues R345-S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, approximately 50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.     

KCNE peptides differently affect voltage sensor equilibrium and equilibration rates in KCNQ1 K+ channels

KCNQ1 voltage-gated K(+) channels assemble with the family of KCNE type I transmembrane peptides to afford membrane-embedded complexes with diverse channel gating properties. KCNQ1/KCNE1 complexes generate the very slowly activating cardiac I(Ks) current, whereas assembly with KCNE3 produces a constitutively conducting complex involved in K(+) recycling in epithelia. To determine whether these two KCNE peptides influence voltage sensing in KCNQ1 channels, we monitored the position of the S4 voltage sensor in KCNQ1/KCNE complexes using cysteine accessibility experiments. A panel of KCNQ1 S4 cysteine mutants was expressed in Xenopus oocytes, treated with the membrane-impermeant cysteine-specific reagent 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and the voltage-dependent accessibility of each mutant was determined. Of these S4 cysteine mutants, three (R228C, G229C, I230C) were modified by MTSET only when KCNQ1 was depolarized. We then employed these state-dependent residues to determine how assembly with KCNE1 and KCNE3 affects KCNQ1 voltage sensor equilibrium and equilibration rates. In the presence of KCNE1, MTSET modification rates for the majority of the cysteine mutants were approximately 10-fold slower, as was recently reported to indicate that the kinetics of the KCNQ1 voltage sensor are slowed by KCNE1 (Nakajo, K., and Y. Kubo. 2007 J. Gen. Physiol. 130:269-281). Since MTS modification rates reflect an amalgam of reagent accessibility, chemical reactivity, and protein conformational changes, we varied the depolarization pulse duration to determine whether KCNE1 slows the equilibration rate of the voltage sensors. Using the state-dependent cysteine mutants, we determined that MTSET modification rates were essentially independent of depolarization pulse duration. These results demonstrate that upon depolarization the voltage sensors reach equilibrium quickly in the presence of KCNE1 and the slow gating of the channel complex is not due to slowly moving voltage sensors. In contrast, all cysteine substitutions in the S4 of KCNQ1/KCNE3 complexes were freely accessible to MTSET independent of voltage, which is consistent with KCNE3 shifting the voltage sensor equilibrium to favor the active state at hyperpolarizing potentials. In total, these results suggest that KCNE peptides differently modulate the voltage sensor in KCNQ1 K(+) channels.     

Side chain orientation in the selectivity filter of a voltage-gated Ca2+ channel

Four glutamate residues (EEEE locus) are essential for ion selectivity in voltage-gated Ca(2+) channels, with ion-specific differences in binding to the locus providing the basis of selectivity. Whether side chain carboxylates or alternatively main chain carbonyls of these glutamates project into the pore to form the ion-binding locus has been uncertain. We have addressed this question by examining effects of sulfhydryl-modifying agents (methanethiosulfonates) on 20 cysteine-substituted mutant forms of an L-type Ca(2+) channel. Sulfhydryl modifiers partially blocked whole oocyte Ba(2+) currents carried by wild type channels, but this block was largely reversed with washout. In contrast, each of the four EEEE locus glutamate --> cysteine mutants (0 position) was persistently blocked by sulfhydryl modifiers, indicating covalent attachment of a modifying group to the side chain of the substituted cysteine. Cysteine substitutions at positions immediately adjacent to the EEEE locus glutamates (+/-1 positions) were also generally susceptible to sulfhydryl modification. Sulfhydryl modifiers had lesser effects on channels substituted one position further from the EEEE locus (+/-2 positions). These results indicate that the carboxylate-bearing side chains of the EEEE locus glutamates and their immediate neighbors project into the water-filled lumen of the pore to form an ion-binding locus. Thus the structure of the Ca(2+) channel selectivity filter differs substantially from that of ancestral K(+) channels.     

Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines

Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current–voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl⁻ permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.     

Cystic fibrosis transmembrane conductance regulator: solution structures of peptides based on the Phe508 region, the most common site of disease-causing DeltaF508 mutation.

Most cases of cystic fibrosis (CF), a common inherited disease of epithelial cell origin, are caused by the deletion of Phe508 located in the first nucleotide-binding domain (NBF1) of the protein called CFTR (cystic fibrosis transmembrane conductance regulator). To gain greater insight into the structure within the Phe508 region of the wild-type protein and the change in structure that occurs when this residue is deleted, we conducted nuclear magnetic resonance (NMR) studies on representative synthetic 26 and 25 amino acid peptide segments. 2D 1H NMR studies at 600 MHz of the 26-residue peptide consisting of Met498 to Ala523 in 10% DMSO, pH 4.0, at 25 degrees C show a continuous but labile helix from Gly500 to Lys522, based on both NH-NH(i,i+1) and alphaH-NH(i,i+1) NOEs. Phe508 within this helix shows only short-range (i, 相似文献   

Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.     

SCAM analysis reveals a discrete region of the pore turret that modulates slow inactivation in Kv1.5

In Kv1.5, protonation of histidine 463 in the S5-P linker (turret) increases the rate of depolarization-induced inactivation and decreases the peak current amplitude. In this study, we examined how amino acid substitutions that altered the physico-chemical properties of the side chain at position 463 affected slow inactivation and then used the substituted cysteine accessibility method (SCAM) to probe the turret region (E456-P468) to determine whether residue 463 was unique in its ability to modulate the macroscopic current. Substitutions at position 463 of small, neutral (H463G and H463A) or large, charged (H463R, H463K, and H463E) side groups accelerated inactivation and induced a dependency of the current amplitude on the external potassium concentration. When cysteine substitutions were made in the distal turret (T462C-P468C), modification with either the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) or negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate reagent irreversibly inhibited current. This inhibition could be antagonized either by the R487V mutation (homologous to T449V in Shaker) or by raising the external potassium concentration, suggesting that current inhibition by MTS reagents resulted from an enhancement of inactivation. These results imply that protonation of residue 463 does not modulate inactivation solely by an electrostatic interaction with residues near the pore mouth, as proposed by others, and that residue 463 is part of a group of residues within the Kv1.5 turret that can modulate P/C-type inactivation. electrophysiology; voltage-gated potassium channels; substituted cysteine accessibility method     

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na⁺ channel (ENaC) is modulated by Na⁺ self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na⁺. A His residue within the extracellular domain of γENaC (γHis²³⁹) was found to have a critical role in Na⁺ self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na⁺ self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na⁺ self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na⁺ self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the γH239F mutant showed that, in the absence of Na⁺ self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that γHis²³⁹ functions within an extracellular allosteric regulatory subdomain of the γ subunit that has an important role in conferring the response of the channel to external Na⁺.     

Conformationally sensitive residues in extracellular loop 5 of the Na+/dicarboxylate co-transporter

The Na+/dicarboxylate co-transporter, NaDC-1, from the kidney and small intestine, transports three sodium ions together with one divalent anion substrate, such as succinate2-. A previous study (Pajor, A. M. (2001) J. Biol. Chem. 276, 29961-29968), identified four amino acids, Ser-478, Ala-480, Ala-481, and Thr-482, near the extracellular end of transmembrane helix (TM) 9 that are likely to form part of the permeation pathway of the transporter. All four cysteine-substituted mutants were sensitive to inhibition by the membrane-impermeant reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) and protected by substrate. In the present study, we continued the cysteine scan through extracellular loop 5 and TM10, from Thr-483 to Val-528. Most cysteine substitutions were well tolerated, although cysteine mutations of some residues, particularly within the TM, produced proteins that were not expressed on the plasma membrane. Six residues in the extracellular loop (Thr-483, Thr-484, Leu-485, Leu-487, Ile-489, and Met-493) were sensitive to chemical labeling by MTSET, depending on the conformational state of the protein. Transport inhibition by MTSET could be prevented by substrate regardless of temperature, suggesting that the likely mechanism of substrate protection is steric hindrance rather than large-scale conformational changes associated with translocation. We conclude that extracellular loop 5 in NaDC-1 appears to have a functional role, and it is likely to be located in or near the substrate translocation pore in the protein. Conformational changes in the protein affect the accessibility of the residues in extracellular loop 5 and provide further evidence of large-scale changes in the structure of NaDC-1 during the transport cycle.     

Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits

Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.