A multipoint hydrogen-bond network underlying KcsA C-type inactivation

In the prokaryotic potassium channel KcsA activation gating at the inner bundle gate is followed by C-type inactivation at the selectivity filter. Entry into the C-type inactivated state has been directly linked to the strength of the H-bond interaction between residues Glu-71 and Asp-80 behind the filter, and is allosterically triggered by the rearrangement of the inner bundle gate. Here, we show that H-bond pairing between residues Trp-67 and Asp-80, conserved in most K⁺ channels, constitutes another critical interaction that determines the rate and extent of KcsA C-type inactivation. Disruption of the equivalent interaction in Shaker (Trp-434-Asp-447) and Kv1.2 (Trp-366-Asp-379) leads also to modulation of the inactivation process, suggesting that these residues also play an analogous role in the inactivation gating of Kv channels. The present results show that in KcsA C-type inactivation gating is governed by a multipoint hydrogen-bond network formed by the triad Trp-67-Glu71-Asp-80. This triad exerts a critical role in the dynamics and conformational stability of the selectivity filter and might serve as a general modulator of selectivity filter gating in other members of the K⁺ channel family.     

Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Concerted gating mechanism underlying KATP channel inhibition by ATP

KATP channels assemble from four regulatory SUR1 and four pore-forming Kir6.2 subunits. At the single-channel current level, ATP-dependent gating transitions between the active burst and the inactive interburst conformations underlie inhibition of the KATP channel by intracellular ATP. Previously, we identified a slow gating mutation, T171A in the Kir6.2 subunit, which dramatically reduces rates of burst to interburst transitions in Kir6.2DeltaC26 channels without SUR1 in the absence of ATP. Here, we constructed all possible mutations at position 171 in Kir6.2DeltaC26 channels without SUR1. Only four substitutions, 171A, 171F, 171H, and 171S, gave rise to functional channels, each increasing Ki,ATP for ATP inhibition by >55-fold and slowing gating to the interburst by >35-fold. Moreover, we investigated the role of individual Kir6.2 subunits in the gating by comparing burst to interburst transition rates of channels constructed from different combinations of slow 171A and fast T171 "wild-type" subunits. The relationship between gating transition rate and number of slow subunits is exponential, which excludes independent gating models where any one subunit is sufficient for inhibition gating. Rather, our results support mechanisms where four ATP sites independently can control a single gate formed by the concerted action of all four Kir6.2 subunit inner helices of the KATP channel.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

Localization of the activation gate of a voltage-gated Ca2+ channel

Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.     

A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na_VAB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629–631 as the narrowest part of the permeation pathway with an estimated pore diameter of <5.8 Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na_VAB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca²⁺ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.     

Thermodynamic coupling between activation and inactivation gating in potassium channels revealed by free energy molecular dynamics simulations

The amount of ionic current flowing through K(+) channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.     

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K⁺ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the “helix-bundle crossing”. However, in the inwardly rectifying (Kir) potassium channel family, the role of this “hinge” residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper “hinge” residues are in close contact with the base of the pore α-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the “lower” gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.     

A gate in the selectivity filter of potassium channels

The selectivity filter of potassium channels is the structural element directly responsible for the selective and rapid conduction of K+, whereas other parts of the protein are thought to function as a molecular gate that either permits or blocks the passage of ions. However, whether the selectivity filter itself also possesses the ability to play the role of a gate is an unresolved question. Using free energy molecular dynamics simulations, it is shown that the reorientation of two peptide linkages in the selectivity filter of the KcsA K+ channel can lead to a stable nonconducting conformational state. Two microscopic factors influence the transition toward such a conformational state: the occupancy of one specific cation binding site in the selectivity filter (S2), and the strength of intersubunit interactions involving the GYG signature sequence. These results suggest that such conformational transitions occurring in the selectivity filter might be related to different K+ channel gating events, including C-type (slow) inactivation.     

Gating of the ATP-sensitive K channel by a pore-lining phenylalanine residue

ATP-sensitive K⁺ (K_ATP) channels are gated by intracellular ATP, proton and phospholipids. The pore-forming Kir6.2 subunit has all essential machineries for channel gating by these ligands. It is known that channel gating involves the inner helix bundle of crossing in which a phenylalanine residue (Phe168) is found in the TM2 at the narrowest region of the ion-conduction pathway in the Kir6.2. Here we present evidence that Phe168-Kir6.2 functions as an ATP- and proton-activated gate via steric hindrance and hydrophobic interactions. Site-specific mutations of Phe168 to a small amino acid resulted in losses of the ATP- and proton-dependent gating, whereas the channel gating was well maintained after mutation to a bulky tryptophan, supporting the steric hindrance effect. The steric hindrance effect, though necessary, was insufficient for the gating, as mutating Phe168 to a bulky hydrophilic residue severely compromised the channel gating. Single-channel kinetics of the F168W mutant resembled the wild-type channel. Small residues increased P_open, and displayed long-lasting closures and long-lasting openings. Kinetic modeling showed that these resulted from stabilization of the channel to open and long-lived closed states, suggesting that a bulky and hydrophobic residue may lower the energy barrier for the switch between channel openings and closures. Thus, it is likely that the Phe168 acts as not only a steric hindrance gate but also potentially a facilitator of gating transitions in the Kir6.2 channel.     

Directional K+ channel insertion in a single phospholipid bilayer: Neutron reflectometry and electrophysiology in the joint exploration of a model membrane functional platform

We investigated the insertion of small potassium (K⁺) channel proteins (Kcv_MA-1D and Kcv_NTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.     

Structure of a Prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels

Voltage-gated sodium channels (Na_Vs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial Na_V (BacNa_V) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of Na_VAe1p, a pore-only BacNa_V derived from Na_VAe1, a BacNa_V from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNa_Vs show that two elements defined by the Na_VAe1p structure, an S6 activation gate position and the cytoplasmic tail “neck”, are central to BacNa_V gating. The structure also reveals the selectivity filter ion entry site, termed the “outer ion” site. Comparison with mammalian voltage-gated calcium channel (Ca_V) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of Ca_V high-affinity calcium binding. Our findings underscore commonalities between BacNa_Vs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.     

Mutations in the S6 Gate Isolate a Late Step in the Activation Pathway and Reduce 4-AP Sensitivity in Shaker Kv Channel

K_v channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.     

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.     

Ligand-dependent linkage of the ATP site to inhibition gate closure in the KATP channel

Major advances have been made on the inhibition gate and ATP site of the K(ir)6.2 subunit of the K(ATP) channel, but little is known about conformational coupling between the two. ATP site mutations dramatically disrupt ATP-dependent gating without effect on ligand-independent gating, observed as interconversions between active burst and inactive interburst conformations in the absence of ATP. This suggests that linkage between site and gate is conditionally dependent on ATP occupancy. We studied all substitutions at position 334 of the ATP site in K(ir)6.2deltaC26 that express in Xenopus oocytes. All substitutions disrupted ATP-dependent gating by 10-fold or more. Only positive-charged arginine or lysine at 334, however, slowed ligand-independent gating from the burst, and this was in some but not all patches. Moreover, the polycationic peptide protamine reversed the slowed gating from the burst of 334R mutant channels, and speeded the slow gating from the burst of wild-type SUR1/K(ir)6.2 in the absence of ATP. Our results support a two-step ligand-dependent linkage mechanism for K(ir)6.2 channels in which ATP-occupied sites function to electrostatically dissociate COOH-terminal domains from the membrane, then as in all K(ir) channels, free COOH-terminal domains and inner M2 helices transit to a lower energy state for gate closure.     

Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels

The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.     

Tail end of the s6 segment: role in permeation in shaker potassium channels

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (P(o,max)) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma). Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb(+) ions for K(+) ions reduces the amplitude of single channel currents and makes gamma insensitive to mutations of Y485. These results suggest that Rb(+) ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6(T) residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.     

Pore structure influences gating properties of the T-type Ca2+ channel alpha1G

The selectivity filter of all known T-type Ca2+ channels is built by an arrangement of two glutamate and two aspartate residues, each one located in the P-loops of domains I-IV of the alpha1 subunit (EEDD locus). The mutations of the aspartate residues to glutamate induce changes in the conduction properties, enhance Cd2+ and proton affinities, and modify the activation curve of the channel. Here we further analyze the role of the selectivity filter in the gating mechanisms of T-type channels by comparing the kinetic properties of the alpha1G subunit (CaV3.1) to those of pore mutants containing aspartate-to-glutamate substitution in domains III (EEED) or IV (EEDE). The change of the extracellular pH induced similar effects on the activation properties of alpha1G and both pore mutants, indicating that the larger affinity of the mutant channels for protons is not the cause of the gating modifications. Both mutants showed alterations in several gating properties with respect to alpha1G, i.e., faster macroscopic inactivation in the voltage range from -10 to 50 mV, positive voltage shift and decrease in the voltage sensitivity of the time constants of activation and deactivation, decrease of the voltage sensitivity of the steady-state inactivation, and faster recovery from inactivation for long repolarization periods. Kinetic modeling suggests that aspartate-to-glutamate mutations in the EEDD locus of alpha1G modify the movement of the gating charges and alter the rate of several gating transitions. These changes are independent of the alterations of the selectivity properties and channel protonation.     

Exploration of the pore structure of a peptide-gated Na+ channel

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.     

Viral potassium channels as a robust model system for studies of membrane–protein interaction

The viral channel Kcv_NTS belongs to the smallest K⁺ channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of Kcv_NTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80 pS), high open-probability (> 50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins—Channels for Cellular Networking.