Search

The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it’s molecular identity and structure remain elusive. The transmembrane channel–like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the “selectivity filter” of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known to play key roles in determining anion permeation properties. Consistent with this proximal arrangement of L102, F337 and T338, different mutations at L102 altered anion selectivity and conductance properties in a way that suggests that this residue plays an important role in determining selectivity filter function, albeit a much lesser role than that of F337. These results suggest an asymmetric three-dimensional arrangement of the key selectivity filter region of the pore, as well as having important implications regarding the molecular mechanism of anion permeation. [ABSTRACT FROM AUTHOR]

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO) ions. Two major effects of Pt(NO) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO) altered blocker effects both on Cl permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO) interaction with a single site within the pore. Experiments at reduced extracellular Cl concentration hinted that Pt(NO) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO) bound in the inner vestibule blocks Cl permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed. [ABSTRACT FROM AUTHOR]

The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal. [ABSTRACT FROM AUTHOR]

The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel comprises 12 transmembrane (TM) α-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore. [ABSTRACT FROM AUTHOR]

Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region. [ABSTRACT FROM AUTHOR]

Abstract: The ankyrin transient receptor potential channel TRPA1 is a sensory neuron-specific channel that is gated by various proalgesic agents such as allyl isothiocyanate (AITC), deep cooling or highly depolarizing voltages. How these disparate stimuli converge on the channel protein to open/close its ion-conducting pore is unknown. We identify several residues within the S6 inner pore-forming region of human TRPA1 that contribute to AITC and voltage-dependent gating. Alanine substitution in the conserved mid-S6 proline (P949A) strongly affected the activation/deactivation and ion permeation. The P949A was functionally restored by substitution with a glycine but not by the introduction of a proline at positions −1, −2 or +1, which indicates that P949 is structurally required for the normal functioning of the TRPA1 channel. Mutation N954A generated a constitutively open phenotype, suggesting a role in stabilizing the closed conformation. Alanine substitutions in the distal GXXXG motif decreased the relative permeability of the channel for Ca2+ and strongly affected its activation/deactivation properties, indicating that the distal G962 stabilizes the open conformation. G958, on the other hand, provides additional tuning leading to decreased channel activity. Together these findings provide functional support for the critical role of the putative inner pore region in controlling the conformational changes that determine the transitions between the open and close states of the TRPA1 channel. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it's molecular identity and structure remain elusive. The transmembrane channel-like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex., Competing Interests: AB, CF No competing interests declared, KS Reviewing editor, eLife

Three deep-pore locations, L312, A313, and A316, were identified in a scanning mutagenesis study of the BK (Ca(2+)-activated, large-conductance K(+)) channel S6 pore, where single aspartate substitutions led to constitutively open mutant channels (L312D, A313D, and A316D). To understand the mechanisms of the constitutive openness of these mutant channels, we individually mutated these three sites into the other 18 amino acids. We found that charged or polar side-chain substitutions at each of the sites resulted in constitutively open mutant BK channels, with high open probability at negative voltages, as well as a loss of voltage and Ca(2+) dependence. Given the fact that multiple pore residues in BK displayed side-chain hydrophilicity-dependent constitutive openness, we propose that BK channel opening involves structural rearrangement of the deep-pore region, where multiple residues undergo conformational changes that may increase the exposure of their side chains to the polar environment of the pore.