Prokaryotic Channel Research Articles

Re-assessing the role of the helix bundle crossing in NaK.

Potassium channels play the main role in shaping action potentials across all living organisms. This function is owed to their ability to open and close in response to stimuli, and thus conduct ions in a selective and regulated fashion. Small prokaryotic potassium channels, such as KcsA, have become model systems for investigations of structure, dynamics and function. These channels are amenable to studies by various biophysical techniques and have been used to obtain insight into the molecular mechanisms of ion channel gating and selectivity. In the case of KcsA, the inner gate is allosterically coupled with the selectivity filter, and several ion conduction mechanisms across the selectivity filter have been proposed. To understand how general these gating and selectivity mechanisms are, we have used solution NMR spectroscopy and solid-supported membrane electrophysiology (SSME) to study another small prokaryotic channel NaK. While NaK maintains a very high degree of structural conservation to potassium channel pores, the conformation of the NaK selectivity filter is different. We show that while NaK still displays allosteric coupling between the selectivity filter and the helix bundle crossing, akin to what was observed for KcsA, the helix bundle crossing remains fully open in both conductive and non-conductive channels. In addition, using SSME we find that ion conduction by NaK shows K+ selectivity in the absence of an electric gradient. These observations contrast with the most recent models proposed by NMR and MD simulations on KcsA and other channels.

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.

As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

The insights into calcium ion selectivity provided by ancestral prokaryotic ion channels.

Prokaryotic channels play an important role in the structural biology of ion channels. At the end of the 20th century, the first structure of a prokaryotic ion channel was revealed. Subsequently, the reporting of structures of various prokaryotic ion channels have provided fundamental insights into the structure of ion channels of higher organisms. Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling. Similar to other proteins, Cavs were predicted to have a prokaryotic counterpart; however, it has taken more than 20 years for one to be identified. The homotetrameric channel obtained from Meiothermus ruber generates the calcium ion specific current, so it is named as CavMr. Its selectivity filter contains a smaller number of negatively charged residues than mutant Cavs generated from other prokaryotic channels. CavMr belonged to a different cluster of phylogenetic trees than canonical prokaryotic cation channels. The glycine residue of the CavMr selectivity filter is a determinant for calcium selectivity. This glycine residue is conserved among eukaryotic Cavs, suggesting that there is a universal mechanism for calcium selectivity. A family of homotetrameric channels has also been identified from eukaryotic unicellular algae, and the investigation of these channels can help to understand the mechanism for ion selection that is conserved from prokaryotes to eukaryotes.

Up-regulation of voltage-gated sodium channels by peptides mimicking S4-S5 linkers reveals a variation of the ligand-receptor mechanism

Prokaryotic NaV channels are tetramers and eukaryotic NaV channels consist of a single subunit containing four domains. Each monomer/domain contains six transmembrane segments (S1-S6), S1-S4 being the voltage-sensor domain and S5-S6 the pore domain. A crystal structure of NaVMs, a prokaryotic NaV channel, suggests that the S4-S5 linker (S4-S5L) interacts with the C-terminus of S6 (S6T) to stabilize the gate in the open state. However, in several voltage-gated potassium channels, using specific S4-S5L-mimicking peptides, we previously demonstrated that S4-S5L/S6T interaction stabilizes the gate in the closed state. Here, we used the same strategy on another prokaryotic NaV channel, NaVSp1, to test whether equivalent peptides stabilize the channel in the open or closed state. A NaVSp1-specific S4-S5L peptide, containing the residues supposed to interact with S6T according to the NaVMs structure, induced both an increase in NaVSp1 current density and a negative shift in the activation curve, consistent with S4-S5L stabilizing the open state. Using this approach on a human NaV channel, hNaV1.4, and testing 12 hNaV1.4 S4-S5L peptides, we identified four activating S4-S5L peptides. These results suggest that, in eukaryotic NaV channels, the S4-S5L of DI, DII and DIII domains allosterically modulate the activation gate and stabilize its open state.

Resonance-driven ion transport and selectivity in prokaryotic ion channels.

Ion channels exhibit a remarkably high accuracy in selecting uniquely its associated type of ion. The mechanisms behind ion selectivity are not well understood. Current explanations build mainly on molecular biology and bioinformatics. Here we propose a simple physical model for ion selectivity based on the driven damped harmonic oscillator (DDHO). The driving force for this oscillator is provided by self-organizing harmonic turbulent structures in the dehydrating ionic flow through the ion channel, namely, oscillating pressure waves in one dimension, and toroidal vortices in two and three dimensions. Density fluctuations caused by these turbulences efficiently transmit their energy to aqua ions that resonate with the driving frequency. Consequently, these release their hydration shell and exit the ion channel as free ions. Existing modeling frameworks do not express the required complex spatiotemporal dynamics, hence we introduce a macroscopic continuum model for ionic dehydration and transport, based on the hydrodynamics of a dissipative ionic flow through an ion channel, subject to electrostatic and amphiphilic interactions. This model combines three classical physical fields: Navier-Stokes equations from hydrodynamics, Gauss's law from Maxwell theory, and the convection-diffusion equation from continuum physics. Numerical experiments with mixtures of chemical species of ions in various degrees of hydration indeed reveal the emergence of strong oscillations in the ionic flow that are instrumental in the efficient dehydration and cause a strong ionic jet into the cell. As such, they provide a powerful engine for the DDHO mechanism. Theoretical predictions of the modeling framework match significantly with empirical patch-clamp data. The DDHO standard response curve defines a unique resonance frequency that depends on the mass and charge of the ion. In this way, the driving oscillations act as a selection mechanism that filters out one specific ion. Application of the DDHO model to real ion data shows that this mechanism indeed clearly distinguishes between chemical species and between aqua and bare ions with a large Mahalanobis distance and high oscillator quality. The DDHO framework helps to understand how SNP mutations can cause severe genetic pathologies as they destroy the geometry of the channel and so alter the resonance peaks of the required ion type.

Bases of Bacterial Sodium Channel Selectivity Among Organic Cations

Hille’s (1971) seminal study of organic cation selectivity of eukaryotic voltage-gated sodium channels showed a sharp size cut-off for ion permeation, such that no ion possessing a methyl group was permeant. Using the prokaryotic channel, NaChBac, we found some similarity and two peculiar differences in the selectivity profiles for small polyatomic cations. First, we identified a diverse group of minimally permeant cations for wildtype NaChBac, ranging in sizes from ammonium to guanidinium and tetramethylammonium; and second, for both ammonium and hydrazinium, the charge-conserving selectivity filter mutation (E191D) yielded substantial increases in relative permeability (PX/PNa). The relative permeabilities varied inversely with relative Kd calculated from 1D Potential of Mean Force profiles (PMFs) for the single cations traversing the channel. Several of the cations bound more strongly than Na+, and hence appear to act as blockers, as well as charge carriers. Consistent with experimental observations, the E191D mutation had little impact on Na+ binding to the selectivity filter, but disrupted the binding of ammonium and hydrazinium, consequently facilitating ion permeation across the NaChBac-like filter. We concluded that for prokaryotic sodium channels, a fine balance among filter size, binding affinity, occupancy, and flexibility seems to contribute to observed functional differences.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10−12 to 10−5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels

ABSTRACTIn the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins. A highly flexible S4-like segment exhibiting Lys instead Arg for these membrane proteins is reported. Sequence analysis indicates that, in addition to this S4-like segment, TolQ proteins also show similarity with specific motifs in S2 and S3 from typical V-sensors. Notably, S3 flexibility profiles from typical VSDs and S3-like in TolQ proteins are also similar. Interestingly, TolQ from early divergent prokaryotes are comparatively more flexible than those in modern counterparts or true V-sensors. Regarding the PD, we also found that 2TM K+-channels in early prokaryotes are considerably more flexible than the ones in modern microbes, and such flexibility is comparable to the one present in CNG channels. Voltage dependence is mainly exhibited in prokaryotic CNG channels whose VSD is rigid whereas the eukaryotic CNG channels are considerably more flexible and poorly V-dependent. The implication of the flexibility present in CNG channels, their sensitivity to cyclic nucleotides and the cation selectivity are discussed. Finally, we generated a structural model of the putative cyclic nucleotide-modulated ion channel, which we coined here as AqK, from the thermophilic bacteria Aquifex aeolicus, one of the earliest diverging prokaryotes known. Overall, our analysis suggests that V-sensors in CNG-like channels were essentially rigid in early prokaryotes but raises the possibility that this module was probably part of a very flexible stator protein of the bacterial flagellum motor complex.

Gates for Conversation in Microbes

Gate keeping has been useful to filter information for dispersal, whether for publication or some mode of communication. The ion channels in various microbes organize the flow of communication. The study of ion channels in bacteria has provided beginner insight to the neuron signalling, though the native role of ion channels in bacteria is yet indefinable. We have tried to summarize the structures of cell membranes bound with ion channels in prokaryotes and few eukaryotes. The signalling was combinedly proposed with information processing.

Interpreting the functional role of a novel interaction motif in prokaryotic sodium channels.

Voltage-gated sodium channels enable the translocation of sodium ions across cell membranes and play crucial roles in electrical signaling by initiating the action potential. In humans, mutations in sodium channels give rise to several neurological and cardiovascular diseases, and hence they are targets for pharmaceutical drug developments. Prokaryotic sodium channel crystal structures have provided detailed views of sodium channels, which by homology have suggested potentially important functionally related structural features in human sodium channels. A new crystal structure of a full-length prokaryotic channel, NavMs, in a conformation we proposed to represent the open, activated state, has revealed a novel interaction motif associated with channel opening. This motif is associated with disease when mutated in human sodium channels and plays an important and dynamic role in our new model for channel activation.

Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel

BackgroundTrimeric intracellular cation (TRIC) channels are crucial for Ca2+ handling in eukaryotes and are involved in K+ uptake in prokaryotes. Recent studies on the representative members of eukaryotic and prokaryotic TRIC channels demonstrated that they form homotrimeric units with the ion-conducting pores contained within each individual monomer.ResultsHere we report detailed insights into the ion- and water-binding sites inside the pore of a TRIC channel from Sulfolobus solfataricus (SsTRIC). Like the mammalian TRIC channels, SsTRIC is permeable to both K+ and Na+ with a slight preference for K+, and is nearly impermeable to Ca2+, Mg2+, or Cl–. In the 2.2-Å resolution K+-bound structure of SsTRIC, ion/water densities have been well resolved inside the pore. At the central region, a filter-like structure is shaped by the kinks on the second and fifth transmembrane helices and two nearby phenylalanine residues. Below the filter, the cytoplasmic vestibule is occluded by a plug-like motif attached to an array of pore-lining charged residues.ConclusionsThe asymmetric filter-like structure at the pore center of SsTRIC might serve as the basis for the channel to bind and select monovalent cations. A Velcro-like plug-pore interacting model has been proposed and suggests a unified framework accounting for the gating mechanisms of prokaryotic and eukaryotic TRIC channels.

Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel.

Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability. Here, we show that docosahexaenoic acid (DHA), a key ω-3 polyunsaturated fatty acid in synaptic membranes, enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC. We determined a 3.25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation. The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces. In this previously unobserved conformation, the extracellular-half of the pore-lining M2 is splayed open, reminiscent of the open conformation, while the intracellular-half is constricted, leading to a loss of both water and permeant ions. These findings, in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes, provide novel mechanistic details of desensitization in pentameric channels.

Engineering prokaryotic channels for control of mammalian tissue excitability.

The ability to directly enhance electrical excitability of human cells is hampered by the lack of methods to efficiently overexpress large mammalian voltage-gated sodium channels (VGSC). Here we describe the use of small prokaryotic sodium channels (BacNav) to create de novo excitable human tissues and augment impaired action potential conduction in vitro. Lentiviral co-expression of specific BacNav orthologues, an inward-rectifying potassium channel, and connexin-43 in primary human fibroblasts from the heart, skin or brain yields actively conducting cells with customizable electrophysiological phenotypes. Engineered fibroblasts (‘E-Fibs') retain stable functional properties following extensive subculture or differentiation into myofibroblasts and rescue conduction slowing in an in vitro model of cardiac interstitial fibrosis. Co-expression of engineered BacNav with endogenous mammalian VGSCs enhances action potential conduction and prevents conduction failure during depolarization by elevated extracellular K+, decoupling or ischaemia. These studies establish the utility of engineered BacNav channels for induction, control and recovery of mammalian tissue excitability.

Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such “Cys-less” receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the “Cys-loop” proline is absolutely conserved, suggesting the more fitting name “Pro loop” for that motif, and “Pro-loop receptors” for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.

Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are activated by the binding of agonists to a site distant from the ion conduction path. These membrane proteins consist of distinct ligand-binding and pore domains that interact via an extended interface. Here, we have investigated the role of residues at this interface for channel activation to define critical interactions that couple conformational changes between the two structural units. By characterizing point mutants of the prokaryotic channels ELIC and GLIC by electrophysiology, X-ray crystallography and isothermal titration calorimetry, we have identified conserved residues that, upon mutation, apparently prevent activation but not ligand binding. The positions of nonactivating mutants cluster at a loop within the extracellular domain connecting β-strands 6 and 7 and at a loop joining the pore-forming helix M2 with M3 where they contribute to a densely packed core of the protein. An ionic interaction in the extracellular domain between the turn connecting β-strands 1 and 2 and a residue at the end of β-strand 10 stabilizes a state of the receptor with high affinity for agonists, whereas contacts of this turn to a conserved proline residue in the M2-M3 loop appear to be less important than previously anticipated. When mapping residues with strong functional phenotype on different channel structures, mutual distances are closer in conducting than in nonconducting conformations, consistent with a potential role of contacts in the stabilization of the open state. Our study has revealed a pattern of interactions that are crucial for the relay of conformational changes from the extracellular domain to the pore region of prokaryotic pentameric ligand-gated ion channels. Due to the strong conservation of the interface, these results are relevant for the entire family.

A Novel Multi-Layer Microfluidic Pipette Aspiration Device for Studying Mechanosensitive Vesicles

Mechanosensitive channel of large conductance (MscL) is a prokaryotic channel that opens due to increased membrane tension. It has been reconstituted in vesicles to study how various biophysical factors influence their mechanical gating properties. In recent years, several theoretical and computational models based on molecular dynamics and continuum mechanics have been developed to understand the underlying mechanisms for the activation of MscLs in vesicles. Experimental approaches for MscL gating studies have focused on cell-by-cell techniques, such as pressure patch clamp, on reconstituted MscL vesicles. Due to the limitation of throughput, such studies are time intensive with low sample numbers. Recently, a microfluidic pipette aspiration array device, based on PDMS multi-layer soft-lithography technique, has been developed in our group to trap and apply mechanical perturbation to single cells in a parallel manner1. This device was used to study the stiffness of human breast cancer cell lines and mechanical gating threshold of reconstituted MscL on infected mammalian cell lines. In this work, we have developed a new device that incorporates a pressure valve and a smaller micropipette dimension to increase trapping efficiency of vesicles. Using this device, we demonstrate stable trapping of single vesicles and expand our efforts to study mechanical gating threshold of reconstituted MscL in vesicles formed by electroformation. Development of novel microtechnology tools that can trap single vesicles and exert tension will have numerous applications to the study of other mechanosensitive channels.[1] Lee & Liu, Lab Chip, 2015, 15, 264-273.

P-54PHYSICOCHEMICAL DETERMINANTS OF ALCOHOL BINDING IN A MODEL LIGAND-GATED ION CHANNEL

Pentameric ligand-gated ion channels are heavily implicated in neurological effects of alcohol, yet high-resolution structural data in this family of receptors are limited. The prokaryotic ligand-gated ion channel GLIC is a potentially valuable model system whose structure …

Disulfide Trapping the GABA-A Receptor Extracellular Beta-5/Beta-5’ Loop

GABAA receptors (GABARs) couple GABA binding to opening of a chloride-conducting channel. The GABA binding site is at β/α-subunit interfaces. In a recent GABAR crystal structure, the β5-β5’ loop reaches across the interface, and juts into the neighboring subunit forming a back lid over the GABA binding site. This extended β5-β5’ loop conformation is not in prokaryotic channels, the acetylcholine-binding protein, or the nicotinic acetylcholine receptor.We used disulfide trapping to probe β5-β5’ loop position and dynamics in α1β2γ2L GABARs. We engineered pairs of cysteines at the β/α GABA-binding site interface: αM111C (β5-β5’ loop) with βS104C (β-strand 5) and α1M113C (β5-β5’ loop) with β2D95C (binding Region A). We expressed single and double mutant α1β2γ2L GABARs in Xenopus oocytes. Initially, we tested for disulfide-bonds by measuring effects of DTT and hydrogen peroxide on GABA-mediated currents from α1M111Cβ2S104Cγ2 and α1M113Cβ2D95Cγ2 receptors. They had no effects. Methanethiosulfonate reagents modified the single cysteine mutants but not the double mutants, indicating the cysteine pairs were linked by disulfides. Single-mutant α1β2S104Cγ2 receptors had reduced GABA efficacy and potency, which were rescued in double-mutant α1M111Cβ2S104Cγ2 receptors, suggesting the cysteines interact. These results indicate the α-subunit β5-β5’ loop extends across the intersubunit interface, near β-strand 5 of the β-subunit. Disulfide linking these two regions had no effect on GABA currents, indicating their relative positions do not change during GABA activation and supporting the idea of a concerted GABA-driven motion in the inner β-sheets of the α- and β-subunits. Linking cysteines in aligned positions at the benzodiazepine-binding α1/γ2-interface (α1S106C and γ2I124C) had similar effects. In contrast, tethering α1M113C (β5-β5’ loop) to β2D95C near Region A of the GABA binding site right-shifted GABA concentration-responses, suggesting this crosslink impairs motions in the site required for GABA action.

Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study.

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼ 102 (∼ 20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.

Heteromultimerization of prokaryotic bacterial cyclic nucleotide-gated (bCNG) ion channels, members of the mechanosensitive channel of small conductance (MscS) superfamily.

Traditionally, prokaryotic channels are thought to exist as homomultimeric assemblies, while many eukaryotic ion channels form complex heteromultimers. Here we demonstrate that bacterial cyclic nucleotide-gated channels likely form heteromultimers in vivo. Heteromultimer formation is indicated through channel modeling, pull-down assays, and real-time polymerase chain reaction analysis. Our observations demonstrate that prokaryotic ion channels can display complex behavior and regulation akin to that of their eukaryotic counterparts.