If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Voltage-gated, sodium ion–selective channels (NaV) generate electrical signals contributing to the upstroke of the action potential in animals. NaVs are also found in bacteria and are members of a larger family of tetrameric voltage-gated channels that includes CaVs, KVs, and NaVs. Prokaryotic NaVs likely emerged from a homotetrameric Ca2+-selective voltage-gated progenerator, and later developed Na+ selectivity independently. The NaV signaling complex in eukaryotes contains auxiliary proteins, termed beta (β) subunits, which are potent modulators of the expression profiles and voltage-gated properties of the NaV pore, but it is unknown whether they can functionally interact with prokaryotic NaV channels. Herein, we report that the eukaryotic NaVβ1-subunit isoform interacts with and enhances the surface expression as well as the voltage-dependent gating properties of the bacterial NaV, NaChBac in Xenopus oocytes. A phylogenetic analysis of the β-subunit gene family proteins confirms that these proteins appeared roughly 420 million years ago and that they have no clear homologues in bacterial phyla. However, a comparison between eukaryotic and bacterial NaV structures highlighted the presence of a conserved fold, which could support interactions with the β-subunit. Our electrophysiological, biochemical, structural, and bioinformatics results suggests that the prerequisites for β-subunit regulation are an evolutionarily stable and intrinsic property of some voltage-gated channels.
drive the upstroke of action potentials and are composed of pore forming α- and auxiliary β-subunits. The α-subunits are large (∼250 kDa) complexes that consist of 24 transmembrane helices that form four homologous (but not identical) domains, termed DI–DIV. These hetero-tetrameric channels are sensitive to changes in transmembrane potential (voltage) and support the complex electrical signaling paradigms that underlie behavior, sensation, contraction, and mobility (
). To date, 10 sodium ion selective α-subunits have been classified in humans. The four domain primary structure of eukaryotic voltage-gated calcium selective channels (CaV) and NaVs is the result of two rounds of gene duplication that occurred in protists in a presumptive CaV (
). In tandem, the emergence of the sodium selectivity and fast (submillisecond) voltage-dependent gating served as functional prerequisites for complex action potential firing and neuronal complexity (
). These 30–36-kDa proteins are comprised of an extracellular amino terminus immunoglobulin-like domain, followed by an extracellular juxtamembrane region, a single transmembrane segment, and an intracellular carboxyl terminus; except for the splice variant β1b, which has a non-conserved carboxyl terminus and lacks a transmembrane segment (
). Related NaVβ-subunits genes are expressed in cardiovascular and nervous tissues where they are part of the V-set immunoglobulin superfamily of cell adhesion molecules that exert non-conduction-related functions such as facilitating cell adhesion and cell migration (
). NaV modulation is also seen in a wide range of α- and NaVβ-subunit combinations, where a prominent effect of the β-subunit is enhancement of current density by promoting trafficking of the α-subunit to the plasma membrane and altering α-subunit voltage-dependent gating (
). The underlying stoichiometry, putative interacting surfaces, and mechanisms of β-subunit modulation of NaVs remains poorly understood. Recently, NaVβ-subunits have been shown to play a role in neuronal excitability through novel interactions with KVs, suggesting NaVβ-subunits may influence the function of channel-types outside of the eukaryotic NaV gene family (
). Structural studies of bacterial NaVs (bNaVs) indicate they are surprisingly similar to eukaryotic voltage-gated cation channels, with a “domain-swapped” architecture of four voltage-sensing domains, which surround a central, gated pore (
). Consistent with this observation, bNaVs are phylogenetically related to eukaryotic NaVs (eNaVs) and CaV, but represent a distinct evolutionary lineage. Specifically, bNaVs are the result of an early differentiation from bacterial KVs that likely occurred before the emergence of eukaryotes, whereas eNaVs appeared more recently. Notably, bNaVs are characterized by a homotetrameric quaternary structure, in contrast to the pseudotetrameric single polypeptide eNaV.
The functional and structural relationships between prokaryotic and eukaryotic channel types are not fully known. For instance, whereas eNaVs and bNaVs are both sodium selective, the mechanisms are markedly different (
). On the other hand, bNaVs show a pharmacological profile similar to that of the eNaV family, an observation that suggests that the channels of these two phyla might share structural similarities that support mechanisms of activation and inactivation (
). Despite potential differences, bNaVs have been the subject of extensive functional and structural studies that have provided key insights into mechanisms of cation recognition, drug binding, voltage-sensing, and channel gating (
Here, we report that NaChBac is modulated by the mammalian voltage-gated sodium channel auxiliary β1-subunit in Xenopus laevis oocytes. β-Subunit modulation of NaChBac was isoform-specific, β1 strongly enhanced current amplitudes by affecting cell-surface expression and the voltage dependence of NaChBac gating, whereas β2 marginally decreased cell-surface expression of the channel resulting in modest sodium current amplitudes. Furthermore, in HEK293T cells, β1 and β3 but not β2, co-purified a co-expressed NaChBac indicating that the functional modulation of NaChBac is the product of the formation of a novel protein complex comprised of a bacterial sodium selective pore and a eukaryotic β-subunit. The reciprocal purification of NaChBaC and co-purification of β1 and β3, but not β2 indicates the specificity of this interaction. An evolutionary analysis of the β-subunit V-set immunoglobulin family pinpoints their emergence in osteichthyes, roughly 420 million years ago, and failed to identify related protein families in bacteria. Modeling analysis of the interaction regions between β1 and NaChBac based on the cryo-EM interaction between electric eel β1 and NaV1.4 indicate a conserved interaction of polar residues in the protein–protein interface between the two α-subunits. In total, the data suggests that the gene family that comprises the sodium channel auxiliary β-subunits may act as broad regulators of electrical signaling through the interaction and modulation of multiple channel types. This functional plurality may arise from the strict evolutionary conservation of specific structural elements found in the voltage-gated ion channels.
Eukaryotic NaVβ-subunits are known to modulate eukaryotic NaV channels (
). To test the possibility that eukaryotic β-subunits may also modulate bNaVs, cRNA encoding NaChBac was co-injected with eukaryotic NaVβ1 and β2 in X. laevis oocytes, an expression system that lacks endogenous β-subunit expression, unlike a majority of mammalian cell lines (
). Co-expression of NaChBac with β1 in Xenopus oocytes significantly increased sodium current amplitudes over NaChBac alone and this trend was sustained over a 50-h time course (Fig. 1, A–C). NaChBac injected alone resulted in currents roughly 36 h post-injection, whereas NaChBac/β1-mediated currents were significantly above background 12 h after injection (Fig. 1A). Saturation of the functional expression was not observed, but NaChBac/β1-mediated currents exceeded the level of reliable voltage clamp after 48 h (Fig. 1A). In contrast, the β2 effect on NaChBac-mediated currents was marginal (Fig. 1, A–C) and small sodium currents were measurable only 36 h post-injection and did not increase over longer incubation time (Fig. 1A).
Evidence of a possible β-subunit interaction with NaChBac, either direct or via a complex, was obtained by affinity purification. β1, β2, and β3 were isolated from protein lysates by a C-terminal His6 tag using a Ni-NTA column. HEK293T cells transfected with NaChBac/β1 and NaChBac/β3 showed association via co-pulldown of NaChBac. This was observed by immunoblotting for a NaChBac N-terminal HA probe that was detected in the elution fraction upon pulldown of the β-subunit, which were identified via the V5 C-terminal tag on the β-subunits. Ni-NTA pulldown of NaChBac/β2 showed pulldown of the β-subunit, observed via a C-terminal HA tag, however, NaChBac was not detected in the elution for NaChBac/β2 or NaChBac only (Fig. 2A). This result was replicated with NaChBac pulldown using Strep-Tactin-Sepharose resin against a N-terminally Twin-Strep-tagged NaChBac, whereby both β1 and β3 co-purified with NaChBac, however, β2 was not observed in the NaChBac purification elution (Fig. 2B). This reciprocal purification suggests either a direct interaction or the composition of a complex that is tightly associated with both NaChBac and the β1/3-subunits. However, the presence of an endogenous β1-subunit expressed in HEK293T cells precluded electrophysiological studies of NaChBac and co-expressed NaVβ-subunits. Furthermore, cell lines that lack β1, such as CHL or COS cells, may contain β1b or β3, the latter being a post-transcriptionally regulated soluble β-subunit that is capable of regulating NaVs (
To determine the possible mechanism of current enhancement by the β1-subunit we assayed the plasma membrane appearance of NaChBac by biotinylation of cell-surface proteins in the presence and absence of β1 or β2 in Xenopus oocytes. NaChBac surface expression was detectable biochemically at the plasma membrane after 24 h post-injection and the total (surface + cytoplasmic) expression was comparable in all three conditions (Fig. 1D). However, the biotinylated (plasma membrane) fraction indicates co-expression of β1 promoted the surface expression of NaChBac, whereas β2 co-expression resulted in a reduction of surface-located channels. Densitometric analysis of the biotinylation data reveals that β1 increases NaChBac surface expression by ∼450%, whereas the β2-subunit reduced expression by ∼30% (Fig. 1D).
Given that the data demonstrated that β1 was able to form a stable protein complex with NaChBac and increase the cell-surface channel density, we next assessed possible effects on voltage-gated properties of NaChBac expressed in Xenopus oocytes. First, a variable length depolarizing pulse was used to minimize the effect of channel inactivation on the tail current. Example traces (Fig. 3A) for NaChBac-, NaChBac/β1-, and NaChBac/β2-mediated currents used for analysis of the steady-state activation popen with the resulting tail-current GV curves (Fig. 3B) are depicted. Co-expression of β1, but not β2, left-shifted and steepened the conductance–voltage relationship (GV), compared with NaChBac alone (Table 1). Second, and similarly, β1 left-shifted the voltage dependence of NaChBac steady-state inactivation, whereas β2 did not (Fig. 3C, Table 2). Last, β1 accelerated the voltage-dependent activation and inactivation kinetics (Fig. 3, D and E).
Table 1eNaVβ-subunits affect the gating kinetics of NaChBac
These results suggest a vestigial conservation between the prokaryotes and eukaryotes α/β-subunit structural interaction interface. This hypothesis raises several fundamental questions: do bacterial counterparts of β-subunits exist? If so, when did β1/3 and β2 begin to functionally diverge? To answer these questions, we performed a bioinformatics analysis aimed at tracing the evolutionary history of these proteins. Whereby, we scoured the comprehensive Uniprot database to identify genes homologous to human β1-subunits in other genomes.
This genome-wide scan identified an extensive set of diverse sequences including, as expected, the genes encoding β2, β3, and β4 isoforms in different organisms and provided some insights into their evolutionary history. First and foremost, all identified β-subunit genes are from eukaryotes and, in particular, metazoa. Second, the branch of the dendrogram containing the β-subunits highlights an interesting hierarchy of sequence similarities: β1 and β3 are closer to each other than to the pair formed by β2 and β4. Thus, it appears that the functional divergence between β1 and β2 predates the gene duplication event that gave rise to β3 and β4. We therefore decided to further investigate the close evolutionary relationship between β1 and β3 by inferring their phylogenetic tree (Fig. 4). We found that β1 and β3 have a relatively recent evolutionary history: these genes are found in euteleostomi (bony vertebrates) but not in chondrichthyes (cartilaginous fishes). This observation indicates that the last common ancestor between β1 and β3 occurred around 420 million years ago, and the absence of β2 in chondrichthyes further confirms that β-subunits were not present before the emergence of euteleostomi and therefore unlikely be present in prokaryotes. Furthermore, this possibility extends to the greater family of V-set proteins (pfam.xfam.org/family/PF07686.15#tabview=tab7) containing the immunoglobulin domain, which is not identified in prokaryotes and is sufficient for β-subunit modulatory effects (
Overall, the evolutionary history of β-subunits and, in particular, the lack of a recognizable prokaryotic homologue suggests that the major structural elements of NaVs involved in the α/β interactions are conserved across kingdoms. Indeed, a conserved structural platform for docking β-subunits would explain the interaction of a human auxiliary subunit with a voltage-gated bacterial channel. To examine this possibility, we analyzed the recently determined structure of the complex between β1 and NaV1.4 from electric eel obtained via cryo-electron microscopy (
). This structural information revealed that, besides the interactions between the immunoglobulin domain of β1 and the extracellular loops of NaV1.4, a large set of residue–residue contacts are present between the transmembrane regions of the two proteins. In particular, the transmembrane helix of β1 shares an extended protein–protein interface with the membrane boundary S0 and transmembrane S2 of NaV1.4 DIII. Importantly, this “bidentate” interaction, involving simultaneous contact with the voltage sensor domain and the extracellular part of the pore, dictates a precise arrangement between these two parts of the channel. Utilizing this structure as a template for the β1–α binding domain, we set out to examine these interaction elements in NaChBac. However, because an experimentally derived structural model of NaChBac is not available, we considered the recently determined structure of the full-length model of NaVMs (
), an algorithm that finds the optimal rotations and translation to minimize the root mean square deviation between corresponding Cα atoms in two proteins. In this case, MISTRAL identifies the two transmembrane regions as structural homologues and returns a remarkably small root mean square deviation value (2.3 Å). This is indicated by the closely superimposed pore domains (Fig. 5, A and B), divergence is mainly from the voltage sensors, with the exception of DIII. Thus, it appears that only DIII, the domain involved in the α/β interaction, has conserved the “ancestral” arrangement with respect to the pore domain.
Having ascertained that the structure of a bNaV can provide an interaction interface for the β-subunits similar to that of eNaVs, we focused our attention on the specific networks of residue–residue interactions between the two proteins. To this end we considered a homology model of NaChBac based on the structural template of NaVAb and subject to multi-μs molecular dynamics simulations (
). The superposition of NaChBac and NaV1.4 reveals a structural match as good as the one observed for NaVMs. Moreover, a peculiar pattern emerges in the side chain chemical identity at the protein–protein interface: there are two clusters of polar residues, one in the middle of the membrane and the other more displaced toward the extracellular compartment that are present in both NaChBac and NaV1.4 that engage in directional interaction with hydrophilic residues on β1 (Fig. 5C). We therefore examined the evolutionary pattern of the polar transmembrane helices as a potential basis for a NaChBac preferential interaction with β1/3 over β2/4.
To address these issues, we analyzed comprehensive multiple sequence alignments of eNaVs and bNaVs containing thousands of genes from all sequenced organisms (
). Surprisingly, the residues of S2 involved in the interaction with β1 are the least conserved, not only in bNaVs, but also in eNaVs (Fig. 6A). The absence of a statistical signal of sequence conservation suggests that the α/β interaction might not be absent in many organisms. An “intermittent” presence of a protein–protein interaction, whereby mutation of one or two residues switches “on” or “off” an interaction in different organisms, is not uncommon in evolution and might be at work also in the case of β-subunits (
). However, despite the poor conservation of the contacting residues, a separate analysis of the sequences encoding for DIII reveals a prevalence of hydrophilic residues at the positions where the polar interactions take place (Fig. 6B). This conservation is, to some extent, present also in bacterial channels but not, for instance, in eukaryotic DII.
Eukaryotic NaVβ-subunits are multifunctional transmembrane proteins that can have numerous modulatory effects on the expression and function of the pore-forming NaVα-subunit. In addition to the well known effects on NaV biophysical characteristics by β-subunits, they play important roles in cellular maintenance as adhesion molecules that interact with cytoskeletal ankyrin and contactin proteins, interactions that can also enhance the functional expression of sodium channels at the cell surface (
). In the present study, the data shows that the eukaryotic NaVβ1-subunit significantly increases the peak current and functional properties of the bacterial channel NaChBac in X. laevis oocytes. By contrast, NaVβ2 modestly influenced the number of functional NaChBac channels at the plasma membrane, with no significant effects on channel function. The results surprisingly mirror β-subunits isoform-specific surface enhancement of cardiac NaVs by the β1-subunit, but not by β2 (
). β1 co-expression induced hyperpolarizing shifts in the voltage dependence of both activation and inactivation of NaChBac. Additionally, this functional regulation was supported via a biochemical pulldown assay in mammalian HEK293T cells whereby heterologously expressed NaChBac co-affinity purified with NaVβ1 and NaVβ3 but not with NaVβ2 the reciprocal purification was also observed whereby NaVβ1 and NaVβ3 co-purified with NaChBac, whereas NaVβ2 did not. Furthermore, co-expression of NaVs with the β1-subunit has been shown to increase the peak of brain sodium current and to shift the voltage dependence of inactivation (
). It is not known for eNaVs if the same underlying interactions and mechanisms are responsible for β-subunit-induced surface membrane enhancement and tuning of voltage gating. Recent cryo-EM evidence shows a direct interaction of the β1 extracellular domain with the NaV voltage-sensing domain, the β1 interacting domain is highly conserved with the β3-subunit and we theorize this to be the interface of not only the β1 but also the β3-subunit interaction with NaChBac observed in our co-purification conditions (
). NaVs have a presumed 1:1 ratio with the β-subunit yet this is believed to interact with a distinct voltage-sensing domain, whereas NaChBac like KVs have multiple identical voltage-sensing domains, making it unclear if the stoichiometry of the NaChBac–β-subunit complex is conserved (
). The amenable nature of bNaV structural studies makes it conceivable that a structure of a bNaV–β-subunit complex is feasible and such studies could shed light on the general mechanism(s) of β-subunit regulation.
The current density arising from the inward flux of sodium ions is related to the total number of channels in the surface membrane, the amount of time that they spend in the open conformation (open probability), and the amount of sodium ions that flow through the open channel (unitary conductance). However, up-regulation of the mammalian sodium current by β1 co-expression in both native settings and in heterologous expression systems can be simply explained by enhanced surface expression (
), similar to the effect here by β1 on NaChBac. Yet, for eNaVs or bNaVs the data cannot distinguish between enhanced forward trafficking, enhanced surface stability, or diminished endocytic and/or lysosomal retrieval. Regardless, the enhanced surface expression of NaChBac by co-expression with β1 may have practical applications in the study of other bNaVs that have shown poor expression profiles in heterologous systems.
The extent to which eNaVs can be modulated by β-subunits can depend strongly on the expression system. One possible cause of this functional variability may be the widespread endogenous expression of NaVβ-subunits in mammalian cell lines (
). Interestingly, NaChBac demonstrates robust expression in HEK293T cells in our hands but these currents were not obviously modulated by either β1 or β2 expression, possibly because of a competing interaction with the expressed channel such protein being expressed in HEK293T and not oocytes may be an endogenous β-subunit (not shown). Thus it is possible that the previous reports of expressed NaVs (
), may have been, in fact, channels associated with endogenous β-subunits.
An evolutionary analysis of the β-subunits family reveals that these auxiliary subunits are part of a large family comprising several myelin-associated proteins. Moreover, the phylogenetic tree of these genes reveals that β1/β3 and β2/β4 form two distinct groups. Strikingly, a recent cryo-EM structure of the complex between NaV1.4 and β1 shows that the part of the channel involved in the protein–protein interaction is structurally superimposable to the corresponding region in NaChBac, even though the individual amino acids involved in the α/β interaction are not conserved across evolution. In total, the electrophysiological, biochemical, structural, and bioinformatics data suggests that the structural prerequisites for β-subunit regulation are an evolutionarily stable, intrinsic property of some voltage-gated channels, including bNaVs, that were present before the emergence of the auxiliary proteins.
Expression of NaChBac and β-subunits in Xenopus oocytes
cRNA of NaChBac, rat β1 and β2 were synthesized in vitro (Ambion, mmessage mmachine, T7) and co-injected into Xenopus oocytes (50 ng of total cRNA/oocyte) with 1:1 ratio of NaChBac and diethyl pyrocarbonate-treated water or with β-subunits. The injected oocytes were kept at 18 °C in oocyte Ringer solution 3 (OR3) containing 50% Leibovitz’s L-15 medium, 10 mm HEPES, 0.5% glutamine, and 0.5% gentamycin, pH 7.6. Electrophysiological measurements were performed at various time points post-injection. Cell-surface biotinylation was performed at 24 h post-injection.
Two-electrode voltage clamp
Voltage-clamped sodium currents were recorded with two microelectrodes using an OC-725C voltage clamp (Warner, Hamden, CT) in a standard Ringers solution (in mm): 116 NaCl, 2 KCl, 1 MgCl2, 0.5 CaCl2, 5 HEPES, pH 7.4. Current recordings were performed at 20–22 °C. Glass microelectrodes had resistances of 0.1–1 megaohm and were backfilled with 3 m KCl. The holding potential was −120 mV in all cases to minimize current rundown. GV relationships were derived by plotting the isochronal tail current amplitudes (the current amplitude measured after stepping from the test potential back to a holding potential of −120 mV) as a function of the depolarizing pulse potential. Depolarizing pulses were delivered as envelope protocols to minimize the contribution of inactivation in GV analysis. Displayed current traces show the full active voltage range of GVs. All data are mean ± S.E.
Biochemical detection of NaChBac surface expression
Biotinylation was performed using 63 oocytes per experimental condition. All biotinylation procedures were performed as previously described (
). Briefly, anti-HA high affinity antibody (Roche Applied Science, 11867423001), directed against an HA peptide on the N terminus of NaChBac was used because the C-terminal HA tag on NaChBac increased the current amplitude and abolished modulatory effects of β1 and β2. Mouse monoclonal antibody directed against α-tubulin (T6199) was purchased from Sigma. Both anti-rat (SAB3700526) and anti-mouse (SAB3700993) horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma. HA-tagged NaChBac was detected by incubating the membrane with 1:1,000 diluted anti-HA at 4 °C overnight and 1:10,000 of anti-rat secondary antibody incubation for 1 h at room temperature. The nitrocellulose membrane was stripped with mild stripping buffer, as described in the Abcam stripping for reprobing protocol. For tubulin detection in each sample, membrane was incubated with anti-tubulin (1:1,000) for 3 h at room temperature, followed by a 1-h room temperature incubation with anti-mouse (1:5,000). ImageJ software (NIH) was used to quantify band densities.
Expression and His tag or Strep-Tactin-Sepharose purification of NaChBac and β-subunits in HEK293T cells
HEK293T in 10-cm plates were transfected with 2.5 μg of NaChBac (N-terminal HA or Twin-Strep Tag) and/or rat β1 (C-terminal V5 and His6), β2 (C-terminal HA and His6), or β3 (C-terminal V5 and His6) plasmid using 15 μl of LipoD293 or Polyjet (SignaGen, SL100668, SL100688) 24 h post-trypsinization at ∼60–70% confluence. Two days post-transfection cells were harvested and washed with PBS containing 1 mm benzamidine and phenylmethylsulfonyl fluoride. Pelleted cells were lysed for 10 min in lysis buffer: 150 mm NaCl, 100 mm Tris-HCl, pH 8.0, 1% Triton X-100, and a mini cOmplete protease inhibitor tablet (Roche Applied Science, 118361530). Removal of cellular debris was accomplished by a 20-min 20,000 × g spin at 4 °C.
His tag purification
Lysate was diluted in half with PBS and imidazole to a final 10 mm imidazole concentration before being loaded onto a 250-μl PerfectPRo Ni-NTA-agarose column (5Prime, 2900510). The column was washed 4 times with 1 ml of PBS + 0.1% Triton X-100 and 20 mm imidazole and eluted with 1 ml of PBS + 0.1% Triton X-100 with 500 mm imidazole and concentrated 50-fold with an Amicon Ultra 10-kDa concentrator (UFC501024). Fractions were loaded onto a 9% polyacrylamide gel (1% total lysate, 1% pulldown fraction or 3% elution for β-blots or 12% elution for NaChBac blots) and run out at 50 V for 30 min, 150 V for 1 h. Protein was transferred to polyvinylidene difluoride (GVS NA, 1212637) at 100 V for 1 h.
Lysate was loaded onto a 100-μl Strep-Tactin-Sepharose column (IBA, 2-1201-002) and flow through 3 times. The column was washed with 23 ml of wash buffer (50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 0.1% Triton X-100) and eluted using wash buffer containing 10 mm desthiobiotin (Sigma, D1411) with 3 × 300-μl elutions, of which the second eluent contained ∼80% of protein. Lysate and elution fraction were loaded onto a 9% polyacrylamide gel (1% total lysate, 6.7% of second elution) and run out at 50 V for 30 min and 150 V for 1 h. Protein was transferred to nitrocellulose (GE Healthcare, 10600002) at 100 V for 1 h. Blots were blocked for 1 h with either 3% cold water fish gelatin (Sigma, G7041) or 5% milk in TBST. Primary antibody 1:1,000 anti-HA (ThermoFisher, 2618) or 1:20,000 anti-V5 (ThermoFisher, R960-25) were incubated overnight at 4 °C in blocking buffer. Primary antibody was washed off with 3 × 10-min TBST (0.1% Tween 20) washes before secondary 1:10,000 anti-mouse IgG1 specific (ThermoFisher, A10551) for HA and 1:10,000 anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-035-003) for 1 h before being washed off with 3 × 10-min TBST washes. Strep-Tactin HRP (Bio-Rad, 1610381) was used according to the manufacturer’s stipulations at 1:20,000. Blots were incubated for 5 min in Clarity ECL solution (Bio-Rad, 1705060) before being imaged. Blots shown are representative of three replicates.
Bioinformatics analysis of β-subunits
A multiple sequence alignment was obtained using first HMMER (
) to scan the Uniprot database (RP35 proteome), using phmmer tool to collect homologous sequences of human β1 and β2 sequences. These were then accurately aligned with MAFFT and redundant sequences with pairwise identity higher than 95% were removed to obtain a first general alignment (
). From the derived phylogenetic tree the branches containing SwissProt-annotated β1 and β3 sequences were extracted and the relative sequence realigned with MAFFT to obtain a more refined tree. All the trees were generated using FastTree (
); the statistical significance of each branch was estimated using the bootstrap approach (a posterior probability higher than 0.7 is considered significant).
S. M., S. L., D. G., V. C., and C. A. A. conceptualization; S. M., S. L., L. L., D. G., and V. C. data curation; S. M., S. L., L. L., D. G., and V. C. formal analysis; S. M., L. L., D. G., and V. C. investigation; S. M., S. L., J. D. L., D. G., and V. C. methodology; S. M., S. L., L. L., V. C., and C. A. A. writing-original draft; S. M., J. D. L., V. C., and C. A. A. writing-review and editing; S. L. and C. A. A. resources; J. D. L. and C. A. A. supervision; D. G. and V. C. software; D. G. and V. C. validation; V. C. and C. A. A. funding acquisition; C. A. A. project administration.
The Membrane Protein Structural Dynamics Consortium was supported by National Institutes of Health Grant GM087519.
This work was supported by National Institutes of Health NIGMS Grant GM122420 (to C. A. A.) and American Heart Association Established Investigator Grant 5EIA22180002 (to C. A. A.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.