The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs.

The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.

A tamoxifen receptor within a voltage-gated sodium channel.

Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.

Polycystin Channel Complexes.

Polycystin subunits can form hetero- and homotetrameric ion channels in the membranes of various compartments of the cell. Homotetrameric polycystin channels are voltage- and calcium-modulated, whereas heterotetrameric versions are proposed to be ligand- or autoproteolytically regulated. Their importance is underscored by variants associated with autosomal dominant polycystic kidney disease and by vital roles in fertilization and embryonic development. The diversity in polycystin assembly and subcellular distribution allows for a multitude of sensory functions by this class of channels. In this review, we highlight their recent structural and functional characterization, which has provided a molecular blueprint to investigate the conformational changes required for channel opening in response to unique stimuli. We consider each polycystin channel type individually, discussing how they contribute to sensory cell biology, as well as their impact on the physiology of various tissues.

Primary cilia TRP channel regulates hippocampal excitability.

Polycystins (PKD2, PKD2L1, and PKD2L2) are members of the transient receptor potential family, which form ciliary ion channels. Most notably, PKD2 dysregulation in the kidney nephron cilia is associated with polycystic kidney disease, but the function of PKD2L1 in neurons is undefined. In this report, we develop animal models to track the expression and subcellular localization of PKD2L1 in the brain. We discover that PKD2L1 localizes and functions as a Ca2+ channel in the primary cilia of hippocampal neurons that apically radiate from the soma. Loss of PKD2L1 expression ablates primary ciliary maturation and attenuates neuronal high-frequency excitability, which precipitates seizure susceptibility and autism spectrum disorder-like behavior in mice. The disproportionate impairment of interneuron excitability suggests that circuit disinhibition underlies the neurophenotypic features of these mice. Our results identify PKD2L1 channels as regulators of hippocampal excitability and the neuronal primary cilia as organelle mediators of brain electrical signaling.

Energetic landscape of polycystin channel gating.

Members of the polycystin family (PKD2 and PKD2L1) of transient receptor potential (TRP) channels conduct Ca2+ and depolarizing monovalent cations. Variants in PKD2 cause autosomal dominant polycystic kidney disease (ADPKD) in humans, whereas loss of PKD2L1 expression causes seizure susceptibility in mice. Understanding structural and functional regulation of these channels will provide the basis for interpreting their molecular dysregulation in disease states. However, the complete structures of polycystins are unresolved, as are the conformational changes regulating their conductive states. To provide a holistic understanding of the polycystin gating cycle, we use computational prediction tools to model missing PKD2L1 structural motifs and evaluate more than 150 mutations in an unbiased mutagenic functional screen of the entire pore module. Our results provide an energetic landscape of the polycystin pore, which enumerates gating sensitive sites and interactions required for opening, inactivation, and subsequent desensitization. These findings identify the external pore helices and specific cross-domain interactions as critical structural regulators controlling the polycystin ion channel conductive and nonconductive states.

Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain.

Genetic variants in PKD2 which encodes for the polycystin-2 ion channel are responsible for many clinical cases of autosomal dominant polycystic kidney disease (ADPKD). Despite our strong understanding of the genetic basis of ADPKD, we do not know how most variants impact channel function. Polycystin-2 is found in organelle membranes, including the primary cilium-an antennae-like structure on the luminal side of the collecting duct. In this study, we focus on the structural and mechanistic regulation of polycystin-2 by its TOP domain-a site with unknown function that is commonly altered by missense variants. We use direct cilia electrophysiology, cryogenic electron microscopy, and superresolution imaging to determine that variants of the TOP domain finger 1 motif destabilizes the channel structure and impairs channel opening without altering cilia localization and channel assembly. Our findings support the channelopathy classification of PKD2 variants associated with ADPKD, where polycystin-2 channel dysregulation in the primary cilia may contribute to cystogenesis.

Disrupting polycystin-2 EF hand Ca2+ affinity does not alter channel function or contribute to polycystic kidney disease.

Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage-dependent gating and subsequent desensitization - two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycystin-2 intracellular EF hand is responsible for these forms of channel regulation, and, if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest that additional Ca2+-binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.

Opening TRPP2 (PKD2L1) requires the transfer of gating charges.

The opening of voltage-gated ion channels is initiated by transfer of gating charges that sense the electric field across the membrane. Although transient receptor potential ion channels (TRP) are members of this family, their opening is not intrinsically linked to membrane potential, and they are generally not considered voltage gated. Here we demonstrate that TRPP2, a member of the polycystin subfamily of TRP channels encoded by the PKD2L1 gene, is an exception to this rule. TRPP2 borrows a biophysical riff from canonical voltage-gated ion channels, using 2 gating charges found in its fourth transmembrane segment (S4) to control its conductive state. Rosetta structural prediction demonstrates that the S4 undergoes ∼3- to 5-Å transitional and lateral movements during depolarization, which are coupled to opening of the channel pore. Here both gating charges form state-dependent cation-π interactions within the voltage sensor domain (VSD) during membrane depolarization. Our data demonstrate that the transfer of a single gating charge per channel subunit is requisite for voltage, temperature, and osmotic swell polymodal gating of TRPP2. Taken together, we find that irrespective of stimuli, TRPP2 channel opening is dependent on activation of its VSDs.

Valproic acid interactions with the NavMs voltage-gated sodium channel.

Valproic acid (VPA) is an anticonvulsant drug that is also used to treat migraines and bipolar disorder. Its proposed biological targets include human voltage-gated sodium channels, among other membrane proteins. We used the prokaryotic NavMs sodium channel, which has been shown to be a good exemplar for drug binding to human sodium channels, to examine the structural and functional interactions of VPA. Thermal melt synchrotron radiation circular dichroism spectroscopic binding studies of the full-length NavMs channel (which includes both pore and voltage sensor domains), and a pore-only construct, undertaken in the presence and absence of VPA, indicated that the drug binds to and destabilizes the channel, but not the pore-only construct. This is in contrast to other antiepileptic compounds that have previously been shown to bind in the central hydrophobic core of the pore region of the channel, and that tend to increase the thermal stability of both pore-only constructs and full-length channels. Molecular docking studies also indicated that the VPA binding site is associated with the voltage sensor, rather than the hydrophobic cavity of the pore domain. Electrophysiological studies show that VPA influences the block and inactivation rates of the NavMs channel, although with lower efficacy than classical channel-blocking compounds. It thus appears that, while VPA is capable of binding to these voltage-gated sodium channels, it has a very different mode and site of action than other anticonvulsant compounds.

Molecular basis of ion permeability in a voltage-gated sodium channel.

Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) â‰« K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.

TRPM7 senses oxidative stress to release Zn2+ from unique intracellular vesicles.

TRPM7 (transient receptor potential cation channel subfamily M member 7) regulates gene expression and stress-induced cytotoxicity and is required in early embryogenesis through organ development. Here, we show that the majority of TRPM7 is localized in abundant intracellular vesicles. These vesicles (M7Vs) are distinct from endosomes, lysosomes, and other familiar vesicles or organelles. M7Vs accumulate Zn2+ in a glutathione-enriched, reduced lumen when cytosolic Zn2+ concentrations are elevated. Treatments that increase reactive oxygen species (ROS) trigger TRPM7-dependent Zn2+ release from the vesicles, whereas reduced glutathione prevents TRPM7-dependent cytosolic Zn2+ influx. These observations strongly support the notion that ROS-mediated TRPM7 activation releases Zn2+ from intracellular vesicles after Zn2+ overload. Like the endoplasmic reticulum, these vesicles are a distributed system for divalent cation uptake and release, but in this case the primary divalent ion is Zn2+ rather than Ca2.

Direct recording and molecular identification of the calcium channel of primary cilia.

A primary cilium is a solitary, slender, non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane. Housing components of the cell division apparatus between cell divisions, primary cilia also serve as specialized compartments for calcium signalling and hedgehog signalling pathways. Specialized sensory cilia such as retinal photoreceptors and olfactory cilia use diverse ion channels. An ion current has been measured from primary cilia of kidney cells, but the responsible genes have not been identified. The polycystin proteins (PC and PKD), identified in linkage studies of polycystic kidney disease, are candidate channels divided into two structural classes: 11-transmembrane proteins (PKD1, PKD1L1 and PKD1L2) remarkable for a large extracellular amino terminus of putative cell adhesion domains and a G-protein-coupled receptor proteolytic site, and the 6-transmembrane channel proteins (PKD2, PKD2L1 and PKD2L2; TRPPs). Evidence indicates that the PKD1 proteins associate with the PKD2 proteins via coiled-coil domains. Here we use a transgenic mouse in which only cilia express a fluorophore and use it to record directly from primary cilia, and demonstrate that PKD1L1 and PKD2L1 form ion channels at high densities in several cell types. In conjunction with an accompanying manuscript, we show that the PKD1L1-PKD2L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established hedgehog pathways.

Primary cilia are specialized calcium signalling organelles.

Primary cilia are solitary, non-motile extensions of the centriole found on nearly all nucleated eukaryotic cells between cell divisions. Only ∼200-300 nm in diameter and a few micrometres long, they are separated from the cytoplasm by the ciliary neck and basal body. Often called sensory cilia, they are thought to receive chemical and mechanical stimuli and initiate specific cellular signal transduction pathways. When activated by a ligand, hedgehog pathway proteins, such as GLI2 and smoothened (SMO), translocate from the cell into the cilium. Mutations in primary ciliary proteins are associated with severe developmental defects. The ionic conditions, permeability of the primary cilia membrane, and effectiveness of the diffusion barriers between the cilia and cell body are unknown. Here we show that cilia are a unique calcium compartment regulated by a heteromeric TRP channel, PKD1L1-PKD2L1, in mice and humans. In contrast to the hypothesis that polycystin (PKD) channels initiate changes in ciliary calcium that are conducted into the cytoplasm, we show that changes in ciliary calcium concentration occur without substantially altering global cytoplasmic calcium. PKD1L1-PKD2L1 acts as a ciliary calcium channel controlling ciliary calcium concentration and thereby modifying SMO-activated GLI2 translocation and GLI1 expression.

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Voltage-gated sodium channels (NaVs) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial NaVs have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial NaVs. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic NaVs NsVBa (nonselective voltage-gated Bacillus alcalophilus) and NaChBac (bacterial sodium channel from Bacillus halodurans) (IC50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of NsVBa, whereas the local anesthetic drug lidocaine was shown to antagonize NsVBa without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of NsVBa, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of NsVBa in one of the deactivated states. In mammalian NaVs, JZTx-27 preferably inhibited the inactivation of NaV1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic NaVs. More important, we proposed that JZTx-27 stabilized the NsVBa VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of NaVs.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel.

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism.

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.

Molecular dynamics of ion transport through the open conformation of a bacterial voltage-gated sodium channel.

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.

Structural basis for gating charge movement in the voltage sensor of a sodium channel.

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6-8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3(10)-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4-S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.

Gating charge interactions with the S1 segment during activation of a Na+ channel voltage sensor.

Voltage-gated Na(+) channels initiate action potentials during electrical signaling in excitable cells. Opening and closing of the pore of voltage-gated ion channels are mechanically linked to voltage-driven outward movement of the positively charged S4 transmembrane segment in their voltage sensors. Disulfide locking of cysteine residues substituted for the outermost T0 and R1 gating-charge positions and a conserved negative charge (E43) at the extracellular end of the S1 segment of the bacterial Na(+) channel NaChBac detects molecular interactions that stabilize the resting state of the voltage sensor and define its conformation. Upon depolarization, the more inward gating charges R2 and R3 engage in these molecular interactions as the S4 segment moves outward to its intermediate and activated states. The R4 gating charge does not disulfide-lock with E43, suggesting an outer limit to its transmembrane movement. These molecular interactions reveal how the S4 gating charges are stabilized in the resting state and how their outward movement is catalyzed by interaction with negatively charged residues to effect pore opening and initiate electrical signaling.

ADPKD variants in the PKD2 pore helix cause structural collapse of the gate and distinct forms of channel dysfunction.

PKD2 is a member of the polycystin subfamily of transient receptor potential (TRP) ion channel subunits which traffic and function in primary cilia organelle membranes. Millions of individuals carry pathogenic genetic variants in PKD2 that cause a life-threatening condition called autosomal dominant polycystic kidney disease (ADPKD). Although ADPKD is a common monogenetic disorder, there is no drug cure or available therapeutics which address the underlying channel dysregulation. Furthermore, the structural and mechanistic impact of most disease-causing variants are uncharacterized. Using direct cilia electrophysiology, cryogenic electron microscopy (cryo-EM), and super resolution imaging, we have discovered mechanistic differences in channel dysregulation caused by three germline missense variants located in PKD2's pore helix 1. Variant C632R reduces protein thermal stability, resulting in impaired channel assembly and abolishes primary cilia trafficking. In contrast, variants F629S and R638C retain native cilia trafficking, but exhibit gating defects. Resolved cryo-EM structures (2.7-3.2Å) of the variants indicate loss of critical pore helix interactions and precipitate allosteric collapse of the channels inner gate. Results demonstrate how ADPKD-causing these mutations have divergent and ranging impacts on PKD2 function, despite their shared structural proximity. These unexpected findings underscore the need for mechanistic characterization of polycystin variants, which may guide rational drug development of ADPKD therapeutics. Regarding polycystin nomenclature: The revised and current IUPHAR/BPS nomenclature creates ambiguity regarding the genetic identity of the polycystin family members of transient receptor potential ion channels (TRPP), especially when cross-referencing manuscripts that describe subunits using the former system 1 . Traditionally, the products of polycystin genes (e.g., PKD2) are referred to as polycystin proteins (e.g., polycystin-2). For simplicity and to prevent confusion, we will refer to the polycystin gene name rather than differentiating gene and protein with separate names- a nomenclature we have recently outlined (Annual Reviews in Physiology, Esarte Palomero et al. 2023) 2.