Nitrate-Dependent Control of Shoot K Homeostasis by the Nitrate Transporter1/Peptide Transporter Family Member NPF7.3/NRT1.5 and the Stelar K+ Outward Rectifier SKOR in Arabidopsis

Root-to-shoot translocation and shoot homeostasis of potassium (K) determine nutrient balance, growth, and stress tolerance of vascular plants. To maintain the cation-anion balance, xylem loading of K⁺ in the roots relies on the concomitant loading of counteranions, like nitrate (NO₃⁻). However, the coregulation of these loading steps is unclear. Here, we show that the bidirectional, low-affinity Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) family member NPF7.3/NRT1.5 is important for the NO₃⁻-dependent K⁺ translocation in Arabidopsis (Arabidopsis thaliana). Lack of NPF7.3/NRT1.5 resulted in K deficiency in shoots under low NO₃⁻ nutrition, whereas the root elemental composition was unchanged. Gene expression data corroborated K deficiency in the nrt1.5-5 shoot, whereas the root responded with a differential expression of genes involved in cation-anion balance. A grafting experiment confirmed that the presence of NPF7.3/NRT1.5 in the root is a prerequisite for proper root-to-shoot translocation of K⁺ under low NO₃⁻ supply. Because the depolarization-activated Stelar K⁺ Outward Rectifier (SKOR) has previously been described as a major contributor for root-to-shoot translocation of K⁺ in Arabidopsis, we addressed the hypothesis that NPF7.3/NRT1.5-mediated NO₃⁻ translocation might affect xylem loading and root-to-shoot K⁺ translocation through SKOR. Indeed, growth of nrt1.5-5 and skor-2 single and double mutants under different K/NO₃⁻ regimes revealed that both proteins contribute to K⁺ translocation from root to shoot. SKOR activity dominates under high NO₃⁻ and low K⁺ supply, whereas NPF7.3/NRT1.5 is required under low NO₃⁻ availability. This study unravels nutritional conditions as a critical factor for the joint activity of SKOR and NPF7.3/NRT1.5 for shoot K homeostasis.The macronutrient potassium (K) is essential for plant growth and development because of its crucial roles in various cellular processes (i.e. regulation of enzyme activities), stabilization of protein synthesis, and neutralization of negative charges. In addition, it is a major component of the cation-anion balance and osmoregulation in plants, thereby influencing cellular turgor, xylem and phloem transport, pH homeostasis, and the setting of membrane potentials (Maathuis, 2009; Marschner, 2012; Sharma et al., 2013). K⁺ uptake and distribution in Arabidopsis (Arabidopsis thaliana) are accomplished by a total of 71 membrane proteins that have been assigned to five gene families: the Shaker and Tandem-Pore K⁺ channels (now also including the inward-rectifier K-like (K_ir-like) channels), the K⁺ uptake permeases (KUP/HAK/KT), the K⁺ transporter (HKT) family, and the cation proton antiporters (CPA; Gierth and Mäser, 2007; Gomez-Porras et al., 2012; Sharma et al., 2013).Root xylem loading is a key step for the delivery of nutrients to the shoot (Poirier et al., 1991; Engels and Marschner, 1992a; Gaymard et al., 1998; Takano et al., 2002; Park et al., 2008). Root-to-shoot translocation of K⁺ is mediated by the voltage-dependent Shaker family K⁺ channel Stelar K⁺ Outward Rectifier (SKOR). The gene is primarily expressed in pericycle and root xylem parenchyma cells, and it is down-regulated upon K shortage and in response to treatments with the phytohormones abscisic acid, cytokinin, and auxin. Such gene expression changes are thought to control K⁺ secretion into the xylem sap and K⁺ reallocation through the phloem to adjust root K⁺ transport activity to K⁺ availability and shoot demand (Pilot et al., 2003). SKOR is activated upon membrane depolarization, and it is in a closed state when the driving force for K⁺ is inwardly directed. It elicits outward K⁺ currents, facilitating the release of the cation from the cells into the xylem. The voltage dependency of the channel is modulated by the external K⁺ concentration to minimize the risk of an undesired K⁺ influx under high K⁺ availability (Johansson et al., 2006). Root-to-shoot K⁺ transfer was strongly reduced in the knockout mutant skor-1, resulting in a decreased shoot K content, whereas the root K content remained unaffected (Gaymard et al., 1998).Root xylem loading is subject to the maintenance of a cation-anion balance, and nitrate (NO₃⁻) is the quantitatively most important anion counterbalancing xylem loading of K⁺ (Engels and Marschner, 1993). Members of the Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) transporter family (NPF) play a prominent role in NO₃⁻ uptake and allocation in Arabidopsis (summarized in Krouk et al., 2010; Wang et al., 2012; and Léran et al., 2014). Two of them have recently been reported to control xylem NO₃⁻ loading and unloading. The low-affinity, pH-dependent bidirectional NO₃⁻ transporter NPF7.3/NRT1.5 (subsequently termed NRT1.5) mediates NO₃⁻ efflux from pericycle cells to the xylem vessels, whereas the low-affinity influx protein NPF7.2/NRT1.8 removes NO₃⁻ from the xylem sap and transfers it into xylem parenchyma cells (Lin et al., 2008; Li et al., 2010; Chen et al., 2012). Accordingly, the expression of both genes is oppositely regulated under various stress conditions (Li et al., 2010). In nrt1.5 mutants, NRT1.8 expression is increased, which is thought to enhance NO₃⁻ reallocation to the root (Chen et al., 2012).The NRT1.5 gene is mainly expressed in root pericycle cells close to the xylem, and the protein localizes to the plasma membrane. In nrt1.5 mutants, less NO₃⁻ is transported from the root to the shoot, and the NO₃⁻ concentration in the xylem sap is reduced. However, root-to-shoot NO₃⁻ transport is not completely abolished in these mutants, indicating the existence of additional xylem-loading activities for NO₃⁻ (Lin et al., 2008; Wang et al., 2012). The recent observation that NPF6.3/NRT1.1/CHL1 and NPF6.2/NRT1.4 are also capable of mediating bidirectional NO₃⁻ transport in Xenopus laevis oocytes might indicate that more NPF family members are contributing to xylem loading with NO₃⁻ (Léran et al., 2013).Electrophysiological studies with NRT1.5-expressing X. laevis oocytes revealed that NO₃⁻ excited an inward current at pH 5.5, which would be expected for a proton-coupled nitrate transporter with a proton to nitrate ratio larger than one (Lin et al., 2008). The inward currents elicited by exposure to nitrate were pH dependent, and Lin et al. (2008) observed that NRT1.5 can also facilitate nitrate efflux when the oocytes were incubated at pH 7.4. Lin et al. (2008) concluded that NRT1.5 can transport nitrate in both directions, presumably through a proton-coupled mechanism. Interestingly, a K⁺ gradient was not sufficient to drive NRT1.5-mediated NO₃⁻ export. However, the determination of root and shoot cation concentrations in the nrt1.5-1 mutant revealed that the amount of K⁺ translocated to the shoot was reduced when NO₃⁻ but not NH₄⁺ was supplied as the N source. Therefore, Lin et al. (2008) suggested a regulatory loop between NO₃⁻ and K⁺ at the xylem loading step.A close relationship between these two nutrients concerning uptake, translocation, recycling, and reduction (of NO₃⁻) has been described in physiological studies since the 1960s (e.g. Ben Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985), but only recently, common components in the NO₃⁻ and K⁺ uptake pathways were identified and led to the first ideas of how such a cross talk might be coordinated on the molecular level. The uptake activity of the K⁺ channel AKT1 as well as the affinity of the NO₃⁻ transporter NPF6.3/NRT1.1/CHL1 are both modulated by the activity of CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE23 (CIPK23), which itself is regulated by CALCINEURIN B-LIKE PROTEIN9 (CBL9) under both deficiencies (Xu et al., 2006; Ho et al., 2009). Yet, the details of this interaction in root K⁺ uptake, the (regulation of) xylem loading with K⁺ and NO₃⁻, and the involvement of SKOR and NRT1.5 in this process are unknown.In this study, we approached this problem by investigating the molecular and physiological responses of Arabidopsis wild-type (Columbia-0 [Col-0]), nrt1.5, and skor transfer DNA (T-DNA) insertion lines to varying NO₃⁻ and K⁺ regimes. The nrt1.5 mutant developed an early senescence phenotype under low NO₃⁻ nutrition, which could be attributed to a reduced K⁺ translocation to the shoot. The assessment of nrt1.5 and skor single- and double-knockout lines disclosed an interplay of the two proteins in the NO₃⁻-dependent control of shoot K homeostasis. The presented data indicate that SKOR mediates K⁺ root-to-shoot translocation under high NO₃⁻ and low K⁺ availability, whereas NRT1.5 is important for K⁺ translocation under low NO₃⁻ availability, irrespective of the K⁺ supply.     

The Halophyte Seashore Paspalum Uses Adaxial Leaf Papillae for Sodium Sequestration

Salinity is a growing issue worldwide, with nearly 30% of arable land predicted to be lost due to soil salinity in the next 30 years. Many grass crops that are vital to sustain the world’s caloric intake are salt sensitive. Studying mechanisms of salt tolerance in halophytic grasses, plants that thrive in salt conditions, may be an effective approach to ultimately improve salt-sensitive grass crops. Seashore paspalum (Paspalum vaginatum) is a halophytic Panicoid grass able to grow in salt concentrations near that of seawater. Despite its widespread cultivation as a sustainable turfgrass, the mechanism underlying its ability to retain high Na⁺ concentrations in photosynthetic tissue while maintaining growth remains unknown. We examined the leaf structure and ion content in P. vaginatum ‘HI10’, which shows increased growth under saline conditions, and Paspalum distichum ‘Spence’, which shows reduced growth under salt, to better understand the superior salt tolerance of cv HI10. A striking difference between cv HI10 and cv Spence was the high steady-state level of K⁺ in cv HI10. Imaging further showed that the adaxial surface of both cv HI10 and cv Spence contained dense costal ridges of papillae. However, these unicellular extensions of the epidermis were significantly larger in cv HI10 than in cv Spence. The cv HI10 papillae were shown to act as Na⁺ sinks when plants were grown under saline conditions. We provide evidence that leaf papillae function as specialized structures for Na⁺ sequestration in P. vaginatum, illustrating a possible path for biotechnological improvement of salt-sensitive Panicoid crops with analogous leaf structures.

About 20% of irrigated land is considered saline, with the amount of saline soils increasing worldwide (Mayak et al., 2004). This is due to increased irrigation in agricultural fields necessitated by more frequent droughts due to climate change. This trend is alarming due to the high salt sensitivity of most crop species that we rely on for vital resources. Yield reduction in crops in saline soils amounts to losses on the order of 12 to 27.3 billion U.S. dollars annually (Qadir et al., 2014). Thus, the improvement of salt tolerance in plants will become key in the coming decades. Breeding salt-tolerant crops is a cost-effective approach to improve growth in saline soils. Although much work has focused on breeding salt-tolerant species, progress in this area has been slow due to the complex genetic and physiological nature of the salt response. Furthermore, most research has been conducted on glycophytic model systems that are salt sensitive (Munns and Gilliham, 2015). Unraveling the salt-tolerance mechanisms in halophytes, species that can complete their life cycle in 200 mm salt concentrations, and transferring these pathways into glycophytes is therefore of great interest (Rajalakshmi and Parida, 2012; Roy and Chakraborty, 2014).Both glycophytes and halophytes have evolved a multitude of salt-tolerance mechanisms, including sodium (Na⁺) exclusion, sequestration, and secretion; osmolyte production; ion homeostasis; and reactive oxygen species (ROS) detoxification (Meng et al., 2018). Often, mechanisms present in glycophytes, such as osmolyte production and Na⁺ exclusion, are utilized in halophytes at higher efficiency (Wyn Jones and Storey, 1981; Grieve and Maas, 1984). However, halophytes also use mechanisms that are absent in glycophytes. Salt sequestration and secretion via salt glands is a halophyte-specific mechanism of coping with salt (Flowers and Colmer, 2008). Salt glands are found in over 50 species in 14 angiosperm families with four subtypes: epidermal bladder cells, complex multicellular glands, bicellular glands, and unicellular glands (Dassanayake and Larkin, 2017). The Poales order contains ∼8% of all halophytes (Flowers et al., 2010) and has therefore been the focus of much salt-gland-focused work (Ceccoli et al., 2015). As salt tolerance has independently evolved >70 times in grass lineages (Bennett et al., 2013), studying these salt sequestering/secreting structures in grasses is an excellent approach to better understand salt tolerance mechanisms in halophytes.Most structural and physiological work on salt glands in grasses has been conducted in the Chloridoideae and Oryzoideae subfamilies. Grasses carry either unicellular or bicellular glands, often referred to as glandular trichomes or microhairs, on the leaf surface (Dassanayake and Larkin, 2017). Microhairs have been observed on the leaf surface in all grass subfamilies except the Pooideae, and have evolved diverse functions including the sequestration or secretion of substances such as callose and heavy metals (Burke et al., 2000; Ceccoli et al., 2015). Unicellular structures on the adaxial leaf side able to secrete salt are only found in the Oryzoideae wild rice species Porteresia coarctata (Flowers et al., 1990; Sengupta and Majumder, 2009). Salt glands in the Chloridoideae are bicellular, consisting of a cap cell and a lower basal cell, both of which are dense in cytoplasm and mitochondria (Ceccoli et al., 2015). The cuticle is thickened above the cap cell in some species, forming a cuticular chamber used for storing secreted salts (Amarasinghe and Watson, 1988). In the Panicoideae, a few cases of Na⁺ secretion have been reported (McWhorter et al., 1995; Ramadan and Flowers, 2004), but to date, no sequestration structures have been identified.The Panicoideae subfamily includes the agronomically important food crops maize (Zea mays) and sorghum (Sorghum bicolor) in addition to the biofuel grasses miscanthus (Miscanthus sinensis), switchgrass (Panicum virgatum), and sugarcane (Saccharum officinarum). One of the most salt-tolerant species in the Panicoideae is the halophyte seashore paspalum (Paspalum vaginatum). It is cultivated as a turfgrass worldwide and derives its popularity from its ability to be irrigated with brackish water. P. vaginatum can survive in salt concentrations near that of seawater (Uddin et al., 2012) and uses osmolyte production, ion homeostasis, and Na⁺ exclusion to cope with salt stress (Peacock and Dudeck, 1985; Lee et al., 2008; Guo et al., 2016). However, its ability to maintain growth while accumulating high levels of Na⁺ in leaf tissue remains perplexing.Here, we studied the leaf structure and Na⁺ sequestration in ‘HI10’, a P. vaginatum cultivar, and ‘Spence’, a Paspalum distichum cultivar. P. vaginatum and P. distichum are closely related (and possibly the same species; Eudy et al., 2017), and constitute group “Disticha” in the tribe Paspaleae. P. distichum is less salt tolerant than P. vaginatum and is typically found in freshwater habitats (Leithead et al., 1971). P. vaginatum and P. distichum therefore represent a useful species pair to study salt tolerance. Furthermore, their salt responses can be compared with those of sorghum, a Panicoid glycophyte. Our main research objective was to identify the phenotypic and physiological factors that contribute to the differential tolerance to salt stress of the two Paspalum spp. cultivars and sorghum ‘BTx623’. We show that both Paspalum species contain dense rows of translucent papillae on the adaxial surface. The papillae are unicellular protrusions from epidermal cells and are much larger in cv HI10 than in cv Spence. We further demonstrate that the papillae sequester Na⁺ under salt stress. This study thus provides evidence of Na⁺ sequestration in specialized leaf-borne organs within the Panicoideae.     

A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes

The evolution of Na⁺-selective four-domain voltage-gated channels (4D-Na_vs) in animals allowed rapid Na⁺-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na⁺-selective voltage-gated channels (BacNa_v), they typically exhibit much slower kinetics than 4D-Na_vs, and are not thought to have crossed the prokaryote–eukaryote boundary. As such, the capacity for rapid Na⁺-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na⁺-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Na_v blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na⁺-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Na_vs. The EukCatB channels therefore represent an independent evolution of fast Na⁺-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

Electrical signals trigger rapid physiological events that underpin an array of fundamental processes in eukaryotes, from contractile amoeboid locomotion (Bingley and Thompson, 1962), to the action potentials of mammalian nerve and muscle cells (Hodgkin and Huxley, 1952). These events are mediated by voltage-gated ion channels (Brunet and Arendt, 2015). In excitable animal cells, Ca²⁺- or Na⁺-selective members of the four-domain voltage-gated cation channel family (4D-Ca_v/Na_v) underpin well-characterized signaling processes (Catterall et al., 2017). The 4D-Ca_v/Na_v family is broadly distributed across eukaryotes, contributing to signaling processes associated with motility in some unicellular protist and microalgal species (Fujiu et al., 2009; Lodh et al., 2016), although these channels are absent from land plants (Edel et al., 2017). It is likely that the ancestral 4D-Ca_v/Na_v channel was Ca²⁺-permeable, with Na⁺-selective channels arising later within the animal lineage (Moran et al., 2015). This shift in ion selectivity represented an important innovation in animals, allowing rapid voltage-driven electrical excitability to be decoupled from intracellular Ca²⁺ signaling processes (Moran et al., 2015).Na⁺-selective voltage-gated channels have not been described in other eukaryotes, although a large family of Na⁺-selective channels (BacNa_v) is present in prokaryotes (Ren et al., 2001; Koishi et al., 2004). BacNa_v are single-domain channels that form homotetramers, resembling the four-domain architecture of 4D-Ca_v/Na_v. Studies of BacNa_v channels have provided considerable insight into the mechanisms of gating and selectivity in voltage-dependent ion channels (Payandeh et al., 2012; Zhang et al., 2012). The range of activation and inactivation kinetics of native BacNa_v are generally slower than observed for 4D-Na_v, suggesting that the concatenation and subsequent differentiation of individual pore-forming subunits may have enabled 4D-Na_v to develop specific properties such as fast inactivation, which is mediated by the conserved intracellular Ile–Phe–Met linker between domains III and IV (Fig. 1A; Irie et al., 2010; Catterall et al., 2017).Open in a separate windowFigure 1.EukCatBs represent a novel class of single-domain channels. A, Schematic diagram of a single-domain EukCatB channel. The voltage-sensing module (S1–S4, blue), including conserved positively charged (++) residues of segment (S4) that responds to changes in membrane potential, is shown. The pore module (S5–S6, red) is also indicated, including the SF motif (Ren et al., 2001). The structure of a 4D-Na_v (showing the SF of rat 4D-Na_v1.4 with canonical “DEKA” locus of Na⁺-selective 4D-Na_v1s) is also displayed (right). The Ile–Phe–Met motif of the fast inactivation gate is indicated (West et al., 1992) B, Maximum likelihood phylogenetic tree of single-domain, voltage-gated channels including BacNa_v and the three distinct classes of EukCat channels (EukCatA–C). Representatives of the specialized family of single-domain Ca²⁺ channels identified in mammalian sperm (CatSpers) are also included. SF for each sequence is shown (right). “Position 0” of the high-field–strength site that is known to be important in determining Na⁺ selectivity (Payandeh et al., 2011), is colored red. Channel sequences selected for functional characterization in this study are shown in bold. EukCatA sequences previously characterized (Helliwell et al., 2019) are also indicated, as is NaChBac channel from B. halodurans (Ren et al., 2001). Maximum likelihood bootstrap values (>70) and Bayesian posterior probabilities (>0.95) are above and below nodes, respectively. Scanning electron micrographs of coccolithophores E. huxleyi (scale bar = 2 μm) and S. apsteinii, (scale bar = 10 μm) are shown.We recently identified several classes of ion channel (EukCats) bearing similarity to BacNa_v in the genomes of eukaryotic phytoplankton. Characterization of EukCatAs found in marine diatoms demonstrated that these voltage-gated channels are nonselective (exhibiting permeability to both Na⁺ and Ca²⁺) and play a role in depolarization-activated Ca²⁺ signaling (Helliwell et al., 2019). Two other distinct classes of single-domain channels (EukCatBs and EukCatCs) were also identified that remain uncharacterized. These channels are present in haptophytes, pelagophytes, and cryptophytes (EukCatBs), as well as dinoflagellates (EukCatCs; Helliwell et al., 2019). Although there is a degree of sequence similarity between the distinct EukCat clades, the relationships between clades are not well resolved, and there is not clear support for a monophyletic origin of EukCats. The diverse classes of EukCats may therefore exhibit significant functional differences. Characterization of these different classes of eukaryote single-domain channels is thus vital to our understanding of eukaryote ion channel structure, function, and evolution, and to our gaining insight into eukaryote membrane physiology more broadly.Notably, EukCatB channels were found in ecologically important coccolithophores, a group of unicellular haptophyte algae that represent major primary producers in marine ecosystems. Coccolithophores are characterized by their ability to produce a cell covering of ornate calcium carbonate platelets (coccoliths; Fig. 1B; Taylor et al., 2017). The calcification process plays an important role in global carbon cycling, with the sinking of coccoliths representing a major flux of carbon to the deep ocean. Patch-clamp studies of coccolithophores indicate several unusual aspects of membrane physiology, such as an inwardly rectifying Cl⁻ conductance and a large outward H⁺ conductance at positive membrane potentials, which may relate to the increased requirement for pH homeostasis associated with intracellular calcification. Here we report that EukCatB channels from two coccolithophore species (Emiliania huxleyi and Scyphosphaera apsteinii) act as very fast Na⁺-selective voltage-gated channels that exhibit many similarities to the 4D-Na_vs, which underpin neuronal signaling in animals. Thus, our findings demonstrate that the capacity for rapid Na⁺-based signaling has evolved in certain photosynthetic eukaryotes, contrary to previous widely held thinking.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

Structure versus function: Are new conformations of pannexin 1 yet to be resolved?

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.

IntroductionInitially, the pannexin field got off to an inauspicious start. Because the three proteins of the pannexin family (Panx1, Panx2, and Panx3) were discovered on the basis of their limited sequence homology to the invertebrate innexin gap junction proteins, it was assumed that they, too, would form gap junctions (Panchin et al., 2000; Bruzzone et al., 2003). However, notwithstanding some disputed science, it soon became clear that the gap junction function of pannexins is not realized (Dahl and Locovei, 2006; Huang et al., 2007; Sosinsky et al., 2011). Gap junction formation by pannexins is moreover prevented by glycosylation of the Panx1 protein (Boassa et al., 2007; Penuela et al., 2007; Boassa et al., 2008). It is conceivable, however, that the nonglycosylated form of Panx1 could allow the docking of oligomers in apposing membranes to each other. Indeed, such docking was observed in cryo-EM preparations of a mutant Panx1, N255A (Ruan et al., 2020). However, because glycosylation of the protein is required for membrane trafficking (Boassa et al., 2007; Penuela et al., 2007), it is unlikely that this process occurs in vivo. This notion is supported by the observation that a glycosylation mutant of Panx1 failed to lead to junctional conductance between paired oocytes, while expression of wtPanx1 followed by glycosidase F treatment before pairing of the cells resulted in gap junction formation, albeit at a much reduced rate as compared with the rate observed with connexins (Boassa et al., 2008). Furthermore, the gap junction function also is unlikely because of theoretical reasons and experimental findings, including expression in single cells, such as erythrocytes (Locovei et al., 2006a), and exclusive expression in the apical membrane of polarized epithelial cells (Ransford et al., 2009; Hanner et al., 2012; Shum et al., 2019). Of the three pannexins, only Panx1 has been studied in detail, and a clear channel function is well established for it. Therefore, this Viewpoint will exclusively deal with Panx1.It is generally assumed that an important function of Panx1 is to form ATP release channels. For example, as of this writing, a PubMed search with “pannexin” and “ATP release” as search terms yielded 287 publications. However, the biophysical evidence for this function is sparse, and the supporting evidence is largely correlative and/or based on pharmacological or genetic interference with ATP release. The ATP release function of Panx1 channels has been contested based on evidence that the channels are highly selective for Cl⁻ and the lack of detectable ATP release under the experimental conditions, i.e., activation by voltage, used in these studies (Ma et al., 2012; Romanov et al., 2012).It was long held that the oligomeric state of the Panx1 channel is that of a hexamer (Penuela et al., 2013; Dahl, 2015; Chiu et al., 2018). Recently, in a <6-mo period, six research groups independently published similar cryo-EM structures of the Panx1 membrane channel (Deng et al., 2020; Jin et al., 2020; Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020). All these papers challenge the view of the oligomeric state of the channel. Instead of the hexameric arrangement of identical Panx1 subunits, the channel appears to be formed by a homomeric heptamer.It is uncontested that the Panx1 channel can operate in a conformation in which the channel exhibits a unitary conductance of <100 pS. However, it has also been reported that, under certain experimental conditions, the channel can exhibit several subconductances with very rare sojourns to a maximal conductance of ∼500 pS (Dahl 2015).Activation of the Panx1 channel can be obtained by various stimuli, some physiological or pathological, others in the form of experimental tools. The majority of the physiological stimuli involve ligands, including ATP, glutamate, α adrenergic agonists, and bradykinin, binding to their cognitive receptors and leading to opening of Panx1 channels and “secondary” ATP release (Dahl 2015, 2018). A nonreversible activation of the channel is induced by cleavage with caspase 3 or 8 (Chekeni et al., 2010), leading to cell death. Experimental stimulation can be obtained in some cells but not in others by increasing the extracellular K⁺ concentration (Silverman et al., 2009; Wang and Dahl 2018; Chiu et al., 2018).Here, we present a point of view on these contested issues in the Panx1 field and attempt to reconcile apparently contradictory data or their interpretations. In addition, we ask to what extent the new structural data support the functional data.The structure of Panx1 channels as revealed by cryo-EMThe excitement for the long-awaited Panx1 structure was rewarded with not one but six publications, all appearing within the first half of 2020 and using cryo-EM to image the channel (Deng et al., 2020; Jin et al., 2020; Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020). Five of the six Panx1 publications showed human Panx1 structures (Deng et al., 2020; Jin et al., 2020; Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020), and two showed frog Panx1 structures (Deng et al., 2020; Michalski et al., 2020). To stabilize the frog Panx1 structure, thus improving the resolution for cryo-EM, Michalski et al. (2020) truncated the C terminus by 71 amino acids and removed 24 amino acids from the intracellular loop between transmembrane (TM) helices 2 and 3; this truncation was the version reported. The other publications reported full-length Panx1 structures, although one used a truncated Panx1 variant (Ruan et al., 2020) and another used a mutation (Jin et al., 2020) as their principal references. Because the C terminus–truncated structure and the (combined) WT structure had the highest overall quality of the several Panx1 structures presented by Ruan et al. (2020), it was used for de novo model building, as the reference structure for analyses, and for discussion. For the same reason, Jin et al. (2020) used the double mutation D376E/D379E, which eliminated the caspase-cleavage site, for their de novo model building. The overall architecture and dimensions of the human and frog Panx1 structures were similar, and the extracellular domains (ECDs) and transmembrane domains (TMDs) were nearly identical, except that the N-terminal helix (NTH) of the frog Panx1 is positioned on the intracellular side and not within the TMD as reported in human Panx1. These structures are becoming an important and critical tool to guide functional experiments and open a new venue to perform molecular dynamics simulations.The most novel finding shared by all the Panx1 structures is that instead of the hexamer hitherto believed to represent the native state of the Panx1 channel, the cryo-EM data show unequivocally a homo-heptamer arranged around a central symmetry axis that constitutes the principal permeation pathway. Panx1’s structural envelope adopts an inverted pail shape in the view parallel to the membrane. The heptameric channel is ∼110 Å long and ∼100 Å wide, with a flat ECD protruding ∼35 Å above the cell membrane and an intracellular domain (ICD) extending ∼35 Å into the cytoplasm with the TMD in between (Fig. 1). Both the N and C termini reside on the cytoplasmic side of the channel. Each TMD has four membrane-spanning helices per protomer. The TMD pore is predominantly lined by hydrophobic amino acids. The NTH is short and lines the TMD pore (human Panx1) with the NTH of one subunit interacting with the TMD of the adjacent subunit. Thus, it has been proposed that the NTH helps to maintain a rigid TMD pore (Ruan et al., 2020). Both the extracellular and intracellular pore entrances are lined with positively charged amino acids, making them favorable for negatively charged cargos such as Cl⁻ and ATP to enter and leave the pore.Open in a separate windowFigure 1.Structural model of Panx1. (A) Cartoon representation of Panx1 WT (PDB accession no. 6wbk) stabilized with detergent. The approximate position of the membrane is indicated by the bars. One protomer of the heptameric assembly is indicated in orange. The lipids resolved in the structure are colored in magenta. It is notable that many of the resolved lipids are found at the interface between subunits. CTH, C-terminal helix; CLH, cytoplasmic linker helix. (B) Overlay of different Panx1 cryo-EM–based structures from different groups (PDB accession no. 6lto = gold, WT hPanx1; PDB accession no. 6wbk = blue, hPanx1 ΔC terminus, ΔN terminus; PDB accession no. 6v6d = lilac, WT hPanx1). The structures diverge very little in the ECD and the membrane domain. However, the variation is larger in the ICD. The approximate positions of F54 and I58 are indicated. (C) Comparison of the extracellular pore. Key residues are annotated using their one-letter abbreviation. All structures are shown as backbone traces except W74, which is part of the constriction ring of the pore. The narrowest constriction is indicated by the arrow.A cap structure is formed on the extracellular side from seven ECDs, one from each subunit. The ECDs are organized into the pore’s most constricted site, thus defining the maximum size of permeable molecules and establishing the extracellular entrance to the TMD. Each ECD contains one helix cross-linked through a disulfide bond to a three-stranded antiparallel β-sheet. The N-terminal end of each subunit helix protrudes inward toward the central pore axis contributing to the constriction site. The key residues involved in the gating of Panx1 are part of the W74-R75-D81 inter-subunit triplet (Fig. 1) from the N-terminal ends of the ECD helices and are proposed to provide rigidity to the extracellular entrance containing positive charges (Deng et al., 2020). This ring lining the wall of the constriction defines a hydrated pore diameter of ∼9 Å (Fig. 1) and is the crux of the argument that the extracellular constriction site is the effective selectivity filter discriminating cargoes on the basis of their charge and size. By contrast, the narrowest point of the pore in the TMD has a hydrated diameter of ∼13 Å.Other inter-subunit interactions that stabilize the pore arrangement in the TMD are (1) the F67-Y79 aromatic–aromatic and Q266-T252 hydrogen bonding in the ECD, (2) the TM helix TM1-TM1 and TM2-TM4 interfaces between adjacent subunits, (3) the N-terminal loop from the neighboring subunit extending into the TMD pore lining, and (4) the resolvable residues of C-terminal helix 3 association with the cytoplasmic linker helices 1 and 2 from the adjacent subunit (Fig. 1; Deng et al., 2020; Michalski et al., 2020). In addition, membrane lipids occupy the crevice between TM3 and an adjacent TM4 at the edge of the channel. Lipid head groups associate with positively charged residues from the N-terminal end of TM3 (K214) and the C-terminal domain of TM4 (R302 and K303; Deng et al., 2020). It may be that the lipid environment influences the assembly and possibly the activation of the Panx1 channel.The pore substantially widens toward the intracellular side. The ICDs extend away from the pore axis, producing a voluminous intracellular vestibule. Two helices in the ICD connect to two TMD helices to form a compact assembly that provides rigidity to the vestibule. The vestibule is slightly larger in the frog Panx1 structure, but the pore architecture is essentially the same as in the human version. The C-terminal segment consisting of ∼70 amino acids, including the caspase cleavage site located immediately before the C terminus, was not visualized in any of the published cryo-EM structures, suggesting that it is intrinsically flexible. C-terminal cleavage removes its autoinhibitory effect without inducing an overall conformational change to the pore (Jin et al., 2020).A novel feature presented by Ruan et al. (2020) is seven narrow side tunnels in the upper ICD running perpendicular to and ending at the pore, like a T-intersection. This tunnel network was proposed to allow passage of small anions because each tunnel contains several positively charged and other polar residues along its length. The flexible NTH-TM1 linker would function as a gate. They hypothesized that these side tunnels facilitate Cl⁻ flux when the pore is blocked by the C terminus residing in the ICD vestibule. Movement or cleavage of the C terminus then would induce the ATP-permeable large-pore conformation. Further structural work is needed to determine whether the C terminus actually resides in the ICD vestibule. One complication with this tunnel hypothesis is that each tunnel has two constriction sites formed by R29 and A33, with diameters of 5.8 and 4.0 Å, respectively, meaning that partial dehydration of a Cl⁻ ion is likely required for it to pass through the tunnel. The possibility of anion tunnels in the Panx1 structure raises the question: could there be multiple open (active) conformations, and does “open” mean open to ATP and Cl⁻ ion or Cl⁻ ion only? Three of the six papers (Michalski et al., 2020; Qu et al., 2020) assume that the Panx1 structure is in the closed or inactive conformation, whereas the other three report an open or active conformation. These discrepancies should be resolved with further work. Interestingly, the overlay of three of these structures, one presumably open and two closed, shows nearly identical conformations of the extracellular constriction of the pore (Fig. 1 C).The structures of Panx1 truncation and mutant variants, as well as its docked inhibitor, carbenoxolone (CBX), illuminate the roles of distinct structural elements. For example, cryo-EM revealed that CBX sits atop the ECD and plugs the W74-R75-D81 ring (Jin et al., 2020; Ruan et al., 2020), supporting pore blocking as its mechanism of Panx1 inhibition. Second, removal of the NTH did not much change the shape of the TMD. This truncation still generated a CBX-sensitive, voltage-dependent current similar to the full-length Panx1 current (Ruan et al., 2020), suggesting that the NTH may play a role in the assembly of the heptamer from the Panx1 protomers rather than being an essential element in the pore conduction pathway. Third, the Panx1 structures obtained in the presence of Ca²⁺ and K⁺ were indistinguishable from the untreated Panx1 structure, suggesting that neither Ca²⁺ nor K⁺ is likely to directly activate the Panx1 channel (Ruan et al., 2020). Fourth, the glycosylation-deficient mutant (N255A) generated gap junction structures as well as hemichannels as determined by cryo-EM (Ruan et al., 2020). The N255A gap junction is formed by two hemichannels docked by the interaction of the extracellular linker 2 on apposing ECDs. Ruan et al. (2020) noted that this Panx1 gap junction likely is not a normal physiological conformation.Comparing the Panx1 structure with other ATP-permeant pore structures offers a blueprint for dissecting pore properties of Panx1. The oligomeric configuration of the ATP-permeant channels does not appear to correlate with pore diameter, in particular when comparing the narrowest extent of the pore (Table 1). Like Panx1, CALHM1 was originally thought to adopt a hexameric configuration (Siebert et al., 2013) until the cryo-EM structure revealed an octameric configuration (Syrjanen et al., 2020). It should be noted that not all connexin channels transport ATP. For example, Cx26, Cx30, Cx43, and Cx46 transport ATP, but Cx32 does not (Harris, 2001; Hansen et al., 2014). An interesting comparison can be made between Panx1 and human Cx31.3. Cx31.3 has a pore with a constriction site diameter of ∼8 Å, similar to Panx1’s constriction of ∼9 Å, and also selectively transports Cl⁻ and ATP (Lee et al., 2020). One difference between the two structures is that the constriction is in the ICD’s NTH for Cx31.3 and in the ECD for Panx1. Lee et al. (2020) pointed out that because Cx31.3’s pore constriction at the cytoplasmic entrance is smaller than the effective hydrated diameter of ATP (∼12 Å; Sabirov and Okada, 2004), the reported conformation would not allow ATP to pass through the pore without substantial conformational changes of the NTH. The same argument could be applied to Panx1’s ECD.Table 1.Comparison of oligomeric configuration with pore constriction diameter