Paul G. DeCaen

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

Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Nav channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Nav 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 NavRh, 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 Ca2+ 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 NavRh is in an ‘inactivated’ conformation. Comparison of NavRh with NavAb reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

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.

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.

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 310-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.

Sequential formation of ion pairs during activation of a sodium channel voltage sensor

Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed “gating charges,” which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltage-dependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models.

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus “locking” the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.

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.

Prokaryotic Navms Channel as a Structural and Functional Model for Eukaryotic Sodium Channel Antagonism.

Significance Many drugs used to treat pain, epilepsy, and cardiac arrhythmias target human voltage-gated sodium-selective channels. Surprisingly, we found that a bacterial voltage-gated sodium channel is also inhibited by many eukaryotic sodium channel antagonists. This bacterial channel was crystallized with several brominated blocker compounds, and the high-resolution structures reveal a common antagonist binding site in the cavity of the pore. Electrophysiology studies of channels with mutations at adjacent residues validate the site. These results suggest that despite millions of years of evolution separating human and bacterial sodium channels, these simple bacterial channels can be a valuable tool for screening and rational design of human drugs. 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.

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.

Role of the C-terminal domain in the structure and function of tetrameric sodium channels

Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.