Voltage-gated sodium, potassium, and calcium channels consist of four voltage-sensing domains (VSDs) that surround a central pore domain and transition from a down state to an up state in response to membrane depolarization. While many types of drugs bind pore domains, the number of organic molecules known to bind VSDs is limited. The Hv1 voltage-gated proton channel is made of two VSDs and does not contain a pore domain, providing a simplified model for studying how small ligands interact with VSDs. Here, we describe a ligand, named HIF, that interacts with the Hv1 VSD in the up and down states. We find that HIF rapidly inhibits proton conduction in the up state by blocking the open channel, as previously described for 2-guanidinobenzimidazole and its derivatives. HIF, however, interacts with a site slowly accessible in the down state. Functional studies and MD simulations suggest that this interaction traps the compound in a narrow pocket lined with charged residues within the VSD intracellular vestibule, which results in slow recovery from inhibition. Our findings point to a “wrench in gears” mechanism whereby side chains within the binding pocket trap the compound as the teeth of interlocking gears. We propose that the use of screening strategies designed to target binding sites with slow accessibility, similar to the one identified here, could lead to the discovery of new ligands capable of interacting with VSDs of other voltage-gated ion channels in the down state.
The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K+ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K+ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd2+ and protons on Arabidopsisthaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd2+ at 300 µM depolarizes the V50 of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V50 by 49 mV. Regulation of KAT1 by Cd2+ is state dependent and consistent with Cd2+ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd2+ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd2+ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K+ channels.
more » « less- NSF-PAR ID:
- 10204397
- Publisher / Repository:
- DOI PREFIX: 10.1085
- Date Published:
- Journal Name:
- Journal of General Physiology
- Volume:
- 153
- Issue:
- 1
- ISSN:
- 0022-1295
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
more » « less
Abstract Epithelial calcium channel TRPV6 is a member of the vanilloid subfamily of TRP channels that is permeable to cations and highly selective to Ca2+; it shows constitutive activity regulated negatively by Ca2+and positively by phosphoinositol and cholesterol lipids. In this review, we describe the molecular structure of TRPV6 and discuss how its structural elements define its unique functional properties. High Ca2+selectivity of TRPV6 originates from the narrow selectivity filter, where Ca2+ions are directly coordinated by a ring of anionic aspartate side chains. Divalent cations Ca2+and Ba2+permeate TRPV6 pore according to the knock‐off mechanism, while tight binding of Gd3+to the aspartate ring blocks the channel and prevents Na+from permeating the pore. The iris‐like channel opening is accompanied by an α‐to‐π helical transition in the pore‐lining transmembrane helix S6. As a result of this transition, the intracellular halves of the S6 helices bend and rotate by about 100 deg, exposing different residues to the channel pore in the open and closed states. Channel opening is also associated with changes in occupancy of the transmembrane domain lipid binding sites. The inhibitor 2‐aminoethoxydiphenyl borate (2‐APB) binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1‐S4 transmembrane helical bundle and shifts open‐closed channel equilibrium towards the closed state by outcompeting lipids critical for activation. Ca2+inhibits TRPV6 via binding to calmodulin (CaM), which mediates Ca2+‐dependent inactivation. The TRPV6‐CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head‐to‐tail arrangement. The CaM C‐terminal lobe plugs the channel through a unique cation‐π interaction by inserting the side chain of lysine K115 into a tetra‐tryptophan cage at the ion channel pore intracellular entrance. Recent studies of TRPV6 structure and function described in this review advance our understanding of the role of this channel in physiology and pathophysiology and inform new therapeutic design.
image -
more » « less
EmrE is an
Escherichia coli multidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pK a ) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pK a values for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pK a values and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm ofE. coli . Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pK a values reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains. -
Transient receptor potential (TRP) proteins are a large family of cation-selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state in TRP channels are still a matter of debate. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed phylogenetic reconstruction, sequence statistics, and structural analysis over a large set of TRP channel genes. Here, we report an exceptionally conserved set of residues. This fingerprint is composed of twelve residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbor the apparent differences in coupling strategies used by TRP subgroups.more » « less
-
more » « less
C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.
