Visualizing, quantifying, and manipulating mitochondrial DNA in vivo.

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.

A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling.

BACKGROUND: Intrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags. RESULTS: We generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane. CONCLUSIONS: This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells.

Exploration of inositol 1,4,5-trisphosphate (IP3) regulated dynamics of N-terminal domain of IP3 receptor reveals early phase molecular events during receptor activation.

Inositol 1, 4, 5-trisphosphate (IP3) binding at the N-terminus (NT) of IP3 receptor (IP3R) allosterically triggers the opening of a Ca2+-conducting pore located ~100 Å away from the IP3-binding core (IBC). However, the precise mechanism of IP3 binding and correlated domain dynamics in the NT that are central to the IP3R activation, remains unknown. Our all-atom molecular dynamics (MD) simulations recapitulate the characteristic twist motion of the suppressor domain (SD) and reveal correlated 'clam closure' dynamics of IBC with IP3-binding, complementing existing suggestions on IP3R activation mechanism. Our study further reveals the existence of inter-domain dynamic correlation in the NT and establishes the SD to be critical for the conformational dynamics of IBC. Also, a tripartite interaction involving Glu283-Arg54-Asp444 at the SD - IBC interface seemed critical for IP3R activation. Intriguingly, during the sub-microsecond long simulation, we observed Arg269 undergoing an SD-dependent flipping of hydrogen bonding between the first and fifth phosphate groups of IP3. This seems to play a major role in determining the IP3 binding affinity of IBC in the presence/absence of the SD. Our study thus provides atomistic details of early molecular events occurring within the NT during and following IP3 binding that lead to channel gating.

Structure and Function of IP3 Receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs), by releasing Ca2+ from the endoplasmic reticulum (ER) of animal cells, allow Ca2+ to be redistributed from the ER to the cytosol or other organelles, and they initiate store-operated Ca2+ entry (SOCE). For all three IP3R subtypes, binding of IP3 primes them to bind Ca2+, which then triggers channel opening. We are now close to understanding the structural basis of IP3R activation. Ca2+-induced Ca2+ release regulated by IP3 allows IP3Rs to regeneratively propagate Ca2+ signals. The smallest of these regenerative events is a Ca2+ puff, which arises from the nearly simultaneous opening of a small cluster of IP3Rs. Ca2+ puffs are the basic building blocks for all IP3-evoked Ca2+ signals, but only some IP3 clusters, namely those parked alongside the ER-plasma membrane junctions where SOCE occurs, are licensed to respond. The location of these licensed IP3Rs may allow them to selectively regulate SOCE.

IP3 Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca2+ to Lysosomes.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) allow extracellular stimuli to redistribute Ca2+ from the ER to cytosol or other organelles. We show, using small interfering RNA (siRNA) and vacuolar H+-ATPase (V-ATPase) inhibitors, that lysosomes sequester Ca2+ released by all IP3R subtypes, but not Ca2+ entering cells through store-operated Ca2+ entry (SOCE). A low-affinity Ca2+ sensor targeted to lysosomal membranes reports large, local increases in cytosolic [Ca2+] during IP3-evoked Ca2+ release, but not during SOCE. Most lysosomes associate with endoplasmic reticulum (ER) and dwell at regions populated by IP3R clusters, but IP3Rs do not assemble ER-lysosome contacts. Increasing lysosomal pH does not immediately prevent Ca2+ uptake, but it causes lysosomes to slowly redistribute and enlarge, reduces their association with IP3Rs, and disrupts Ca2+ exchange with ER. In a "piston-like" fashion, ER concentrates cytosolic Ca2+ and delivers it, through large-conductance IP3Rs, to a low-affinity lysosomal uptake system. The involvement of IP3Rs allows extracellular stimuli to regulate Ca2+ exchange between the ER and lysosomes.

Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.

IP3 receptors (IP3Rs) release Ca2+ from the ER when they bind IP3 and Ca2+. The spatial organization of IP3Rs determines both the propagation of Ca2+ signals between IP3Rs and the selective regulation of cellular responses. Here we use gene editing to fluorescently tag endogenous IP3Rs, and super-resolution microscopy to determine the geography of IP3Rs and Ca2+ signals within living cells. We show that native IP3Rs cluster within ER membranes. Most IP3R clusters are mobile, moved by diffusion and microtubule motors. Ca2+ signals are generated by a small population of immobile IP3Rs. These IP3Rs are licensed to respond, but they do not readily mix with mobile IP3Rs. The licensed IP3Rs reside alongside ER-plasma membrane junctions where STIM1, which regulates store-operated Ca2+ entry, accumulates after depletion of Ca2+ stores. IP3Rs tethered close to ER-plasma membrane junctions are licensed to respond and optimally placed to be activated by endogenous IP3 and to regulate Ca2+ entry.

Cyclic AMP Recruits a Discrete Intracellular Ca2+ Store by Unmasking Hypersensitive IP3 Receptors.

Inositol 1,4,5-trisphosphate (IP3) stimulates Ca2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3',5'-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.

Inositol 1,4,5-trisphosphate receptors and their protein partners as signalling hubs.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are expressed in nearly all animal cells, where they mediate the release of Ca(2+) from intracellular stores. The complex spatial and temporal organization of the ensuing intracellular Ca(2+) signals allows selective regulation of diverse physiological responses. Interactions of IP3 Rs with other proteins contribute to the specificity and speed of Ca(2+) signalling pathways, and to their capacity to integrate information from other signalling pathways. In this review, we provide a comprehensive survey of the proteins proposed to interact with IP3 Rs and the functional effects that these interactions produce. Interacting proteins can determine the activity of IP3 Rs, facilitate their regulation by multiple signalling pathways and direct the Ca(2+) that they release to specific targets. We suggest that IP3 Rs function as signalling hubs through which diverse inputs are processed and then emerge as cytosolic Ca(2+) signals.

Fluorescence methods for analysis of interactions between Ca(2+) signaling, lysosomes, and endoplasmic reticulum.

The endoplasmic reticulum (ER) is both the major source of intracellular Ca(2+) for cell signaling and the organelle that forms the most extensive contacts with the plasma membrane and other organelles. Lysosomes fulfill important roles in degrading cellular materials and in cholesterol handling, but they also contribute to Ca(2+) signaling by both releasing and sequestering Ca(2+). Interactions between ER and other Ca(2+)-transporting membranes, notably mitochondria and the plasma membrane, often occur at sites where the two membranes are closely apposed, allowing local Ca(2+) signaling between them. These interactions are often facilitated by scaffold proteins. Recent evidence suggests similar local interactions between ER and lysosomes. We describe simple fluorescence-based methods that allow the interplay between Ca(2+) signals, the ER, and lysosomes to be examined.

Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration.

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca(2+) content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

Preferential assembly of heteromeric small conductance calcium-activated potassium channels.

The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits.

Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum.

Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 µM (LAR-GECO1) and 12 µM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.

Structural organization of signalling to and from IP3 receptors.

In the 30 years since IP3 (inositol 1,4,5-trisphosphate) was first shown to release Ca2+ from intracellular stores, the importance of spatially organized interactions within IP3-regulated signalling pathways has been universally recognized. Recent evidence that addresses three different levels of the structural determinants of IP3-evoked Ca2+ signalling is described in the present review. High-resolution structures of the N-terminal region of the IP3R (IP3 receptor) have established that the two essential phosphate groups of IP3 bind to opposite sides of the IP3-binding site, pulling its two domains together. This conformational change is proposed to disrupt an interaction between adjacent subunits within the tetrameric IP3R that normally holds the channel in a closed state. Similar structural changes are thought to allow gating of ryanodine receptors. cAMP increases the sensitivity of IP3Rs and thereby potentiates the Ca2+ signals evoked by receptors that stimulate IP3 formation. We speculate that both IP3 and cAMP are delivered to IP3Rs within signalling junctions, wherein the associated IP3Rs are exposed to a saturating concentration of either messenger. The concentration-dependent effects of extracellular stimuli come from recruitment of junctions rather than from a graded increase in the activity of individual junctions. IP3Rs within 'IP3 junctions' respond directly to receptors that stimulate phospholipase C, whereas extra-junctional IP3Rs are exposed to suboptimal concentrations of IP3 and open only when they are sensitized by cAMP. These results highlight the importance of selective delivery of diffusible messengers to IP3Rs. The spatial organization of IP3Rs also allows them to direct Ca2+ to specific intracellular targets that include other IP3Rs, mitochondria and Ca2+-regulated channels and enzymes. IP3Rs also interact functionally with lysosomes because Ca2+ released by IP3Rs, but not that entering cells via store-operated Ca2+ entry pathways, is selectively accumulated by lysosomes. This Ca2+ uptake shapes the Ca2+ signals evoked by IP3 and it may regulate lysosomal behaviour.

hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2.

Golgi antiapoptotic proteins (GAAPs) are highly conserved Golgi membrane proteins that inhibit apoptosis and promote Ca(2+) release from intracellular stores. Given the role of Ca(2+) in controlling cell adhesion and motility, we hypothesized that human GAAP (hGAAP) might influence these events. In this paper, we present evidence that hGAAP increased cell adhesion, spreading, and migration in a manner that depended on the C-terminal domain of hGAAP. We show that hGAAP increased store-operated Ca(2+) entry and thereby the activity of calpain at newly forming protrusions. These hGAAP-dependent effects regulated focal adhesion dynamics and cell migration. Indeed, inhibition or knockdown of calpain 2 abrogated the effects of hGAAP on cell spreading and migration. Our data reveal that hGAAP is a novel regulator of focal adhesion dynamics, cell adhesion, and migration by controlling localized Ca(2+)-dependent activation of calpain.

Identification and analysis of putative homologues of mechanosensitive channels in pathogenic protozoa.

Mechanosensitive channels play important roles in the physiology of many organisms, and their dysfunction can affect cell survival. This suggests that they might be therapeutic targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, dysentery, leishmaniasis and trypanosomiasis that are responsible for millions of deaths each year worldwide. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of mechanosensitive channels. Entamoeba histolytica, Leishmania spp., Trypanosoma cruzi and Trichomonas vaginalis have genes encoding homologues of Piezo channels, while most pathogenic protozoa have genes encoding homologues of mechanosensitive small-conductance (MscS) and K(+)-dependent (MscK) channels. In contrast, all parasites examined lack genes encoding mechanosensitive large-conductance (MscL), mini-conductance (MscM) and degenerin/epithelial Na(+) (DEG/ENaC) channels. Multiple sequence alignments of evolutionarily distant protozoan, amoeban, plant, insect and vertebrate Piezo channel subunits define an absolutely conserved motif that may be involved in channel conductance or gating. MscS channels are not present in humans, and the sequences of protozoan and human homologues of Piezo channels differ substantially. This suggests the possibility for specific targeting of mechanosensitive channels of pathogens by therapeutic drugs.

A bead aggregation assay for detection of low-affinity protein-protein interactions reveals interactions between N-terminal domains of inositol 1,4,5-trisphosphate receptors.

Interactions between proteins are a hallmark of all cellular activities. Such interactions often occur with low affinity, a feature that allows them to be rapidly reversible, but it makes them difficult to detect using conventional methods such as yeast 2-hybrid analyses, co-immunoprecipitation or analytical ultracentrifugation. We developed a simple and economical bead aggregation assay to study low-affinity interactions between proteins. By coating beads with interacting proteins, the weak interactions between many proteins are sufficient to allow stable aggregation of beads, an avidity effect. The aggregation is easily measured to allow quantification of protein-protein interactions under a variety of controlled conditions. We use this assay to demonstrate low-affinity interactions between the N-terminal domains of an intracellular Ca(2+) channel, the type 1 inositol 1,4,5-trisphosphate receptor. This simple bead aggregation assay may have widespread application in the study of low-affinity interactions between macromolecules.

Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium.

Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca(2+) content of intracellular stores, and regulate Ca(2+) fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca(2+)-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca(2+) stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum.

Most intracellular Ca(2+) signals result from opening of Ca(2+) channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca(2+) into mitochondria. Ca(2+) channels in lysosomes contribute to endo-lysosomal trafficking and Ca(2+) signalling, but the role of lysosomal Ca(2+) uptake in Ca(2+) signalling is unexplored. Inhibition of lysosomal Ca(2+) uptake by dissipating the H(+) gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca(2+) signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation. Bafilomycin A1 amplified the Ca(2+) signals evoked by photolysis of caged Ins(1,4,5)P(3) or by inhibition of ER Ca(2+) pumps, and it slowed recovery from them. Ca(2+) signals evoked by store-operated Ca(2+) entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca(2+) released by Ins(1,4,5)P(3) receptors.

Identification and analysis of cation channel homologues in human pathogenic fungi.

Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+)), calcium (Ca(2+)) and transient receptor potential (Trp) channels, but not sodium (Na(+)) channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+) and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v) channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+) channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+) channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+) uniporter (MCU). In contrast to humans, which express many K(+), Ca(2+) and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+), Ca(2+) and Trp channel homologues. Furthermore, the sequences of fungal K(+), Ca(2+), Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

Structural changes during HCN channel gating defined by high affinity metal bridges.

Hyperpolarization-activated cyclic nucleotide-sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4-S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd(2+)-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4-S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4-S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4-S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.