Coupling of ATP Hydrolysis with Channel Gating by Purified, Reconstituted CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel situated on the apical membrane of epithelial cells. Our recent studies of purified, reconstituted CFTR revealed that it also functions as an ATPase and that there may be coupling between ATP hydrolysis and channel gating. Both the ATP turnover rate and channel gating are slow, in the range of 0.2 to 1 s^–1, and both activities are suppressed in a disease-causing mutation situated in a putative nucleotide binding motif. Our future studies using purified protein will be directed toward understanding the structural basis and mechanism for coupling between hydrolysis and channel function.     

Interconverting Gating Modes of a Nonselective Cation Channel from the Tapeworm Echinococcus granulosus Reconstituted on Planar Lipid Bilayers

A 107-pS (symmetrical 150 mm KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>0.95) irrespective of either the applied voltage, Ca²⁺, Ba²⁺, or tetraethylammonium concentration. Nevertheless, in contrast with this ``leaklike' behavior, less frequently this ``all-the-time-open' channel reversibly entered two different kinetic modes. One of them was characterized by lower P _o values and some voltage sensitivity (V _?≅ 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a P _o around 0.6–0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms. Received: 30 August 1996/Revised: 31 January 1997     

Stochastic Gating as a Novel Mechanism for Channel Selectivity

An ideal channel, responsible for metabolite fluxes in and out of the cells and cellular compartments, is supposed to be selective for a particular set of molecules only. However, such a channel has to be wide enough to accommodate relatively large metabolites, and, therefore, it allows passage of smaller solutes, for example, sodium, potassium, and chloride ions, thus compromising membrane’s barrier function. Here we show that stochastic gating is able to provide a mechanism for the selectivity of wide channels in favor of large metabolites. Specifically, applying our recent theory of the stochastic gating effect on channel-facilitated transport, we demonstrate that under certain conditions gating hinders translocation of fast-diffusing small solutes to a significantly higher degree than that of large solutes that diffuse much slower. We hypothesize that this can be used by Nature to minimize the shunting effect of wide channels with respect to small solutes.     

Glycosylation Influences Voltage-Dependent Gating of Cardiac and Skeletal Muscle Sodium Channels

The role of glycosylation on voltage-dependent channel gating for the cloned human cardiac sodium channel (hH1a) and the adult rat skeletal muscle isoform (μl) was investigated in HEK293 cells transiently transfected with either hH1a or μl cDNA. The contribution of sugar residues to channel gating was examined in transfected cells pretreated with various glycosidase and enzyme inhibitors to deglycosylate channel proteins. Pretreating transfected cells with enzyme inhibitors castanospermine and swainsonine, or exo-glycosidase neuroaminidase caused 7 to 9 mV depolarizing shifts of V _1/2 for steady-state activation of hH1a, while deglycosylation with corresponding drugs elicited about the same amount of depolarizing shifts (8 to 9 mV) of V _1/2 for steady-state activation of μl. Elevated concentrations of extracellular Mg²⁺ significantly masked the castanospermine-elicited depolarizing shifts of V _1/2 for steady-state activation in both transfected hH1a and μl. For steady-state activation, deglycosylation induced depolarizing shifts of V _1/2 for hH1a (10.6 to 12 mV), but hyperpolarizing shifts for μl (3.6 to 4.4 mV). Pretreatment with neuraminidase had no significant effects on single-channel conductance, the mean open time, and the open probability. These data suggest that glycosylation differentially regulates Na channel function in heart and skeletal muscle myocytes. Received: 8 April 1999/Revised: 18 June 1999     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Gating and Conduction Properties of a Sodium-activated Cation Channel from Lobster Olfactory Receptor Neurons

The gating and conduction properties of a channel activated by intracellular Na⁺ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na⁺. At 210 mm Na⁺, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na⁺. The open time constant was not affected by the concentration of Na⁺, but was increased by membrane depolarization. At 180 mm Na⁺ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na⁺. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na⁺, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na⁺ (P _X /P _Na ), calculated from the reversal potential, was: Li⁺ (1.11) > Na⁺ (1.0) > K⁺ (0.54) > Rb⁺ (0.36) > Cs⁺ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na⁺ current at −60 mV according to the following sequence: Mn²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺ > Ba²⁺. Relative permeabilities for divalent cations (P _Y /P _Na ) were Ca²⁺ (39.0) > Mg²⁺ (34.1) > Mn²⁺ (15.5) > Ba²⁺ (13.8) > Na⁺ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na⁺ and Cs⁺ or Li⁺ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons. Received: 17 September 1996/Revised: 15 November 1996     

Sodium Ions Do Not Stabilize the Selectivity Filter of a Potassium Channel

Ion conduction is an essential function for electrical activity in all organisms. The non-selective ion channel NaK was previously shown to adopt two stable conformations of the selectivity filter. Here, we present solid-state NMR measurements of NaK demonstrating a population shift between these conformations induced by changing the ions in the sample while the overall structure of NaK is not affected. We show that two K⁺-selective mutants (NaK2K and NaK2K-Y66F) suffer a complete loss of selectivity filter stability under Na⁺ conditions, but do not collapse into a defined structure. Widespread chemical shift perturbations are seen between the Na⁺ and K⁺ states of the K⁺-selective mutants in the region of the pore helix indicating structural changes. We conclude that the stronger link between the selectivity filter and the pore helix in the K⁺-selective mutants, compared to the non-selective wild-type NaK channel, reduces the ion-dependent conformational flexibility of the selectivity filter.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

Sodium Channel Fragments: Contributions to Voltage Sensitivity and Ion Selectivity

The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel. Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichroism spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader with repeat III. These structural and functional correlates suggest alternative gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-recognition between the four P-regions is a key component of intact sodium channel selectivity. In addition, the P-region from domain III is significantly voltage-sensitive and molecular dynamics simulations show that the C-terminal part of P-regions is mainly helical whilst the N-terminus tends to unfold. Such specializations of the four domains both in gating and selectivity are independently confirmed in recent electrophysiological studies.     

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high-molecular-weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS-polyacrylamide gels. The apparent molecular weight of the center of the band varies from ～230,000 on 13% acrylamide gels to ～130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.     

Role of Ion Distribution and Energy Barriers for Concerted Motion of Subunits in Selectivity Filter Gating of a K+ Channel

Most potassium channels have two main gate locations, hosting an inner gate at the cytosolic entrance and a filter gate in the selectivity filter; the function of these gates is in many channels coupled. To obtain exclusive insights into the molecular mechanisms that determine opening and closing of the filter gate, we use a combination of single-channel recordings and gating analysis in the minimal viral channel Kcv_NTS. This channel has no inner gate, and its fast closing at negative voltages can therefore be entirely assigned to the filter gate. We find that mutations of S42 in the pore helix severely slow down closing of this filter gate, an effect which is not correlated with hydrogen bond formation by the amino acid at this position. Hence, different from KcsA, which contains the critical E71 in the equivalent position forming a salt bridge, the coupling between selectivity filter and surrounding structures for filter gating must in Kcv_NTS rely on different modes of interaction. Quantitative analysis of concatemers carrying different numbers of S42T mutations reveals that each subunit contributes the same amount of ～ 0.4 kcal/mol to the energy barrier for filter closure indicating a concerted action of the subunits. Since the mutations have neither an influence on the unitary current nor on the voltage dependency of the gate, the data stress that the high subunit cooperativity is mediated through conformational changes rather than through changes in the ion occupation in the selectivity filter.     

Definition of Epitopes for Monoclonal Antibodies Developed Against Purified Sodium Channel Protein: Implications for Channel Structure

To test sodium channel structural models, we defined the epitopes for nineteen independently cloned monoclonal antibodies previously generated against purified, detergent-solubilized, adult rat skeletal muscle sodium channel protein using channel proteolysis, synthetic peptides, and fusion proteins. All identified epitopes were continuous and unique to the skeletal muscle subtype α-subunit. Of the nineteen independent clones, seventeen had epitopes located either in the origin of the amino-terminus or in the interdomain 2–3 region while only two antibodies had epitopes located in the mid-portion of the interdomain 1–2 region. No immunogenic regions were identified on the α-subunit's extracellular regions, interdomain 3–4 segment, or carboxyl-terminus or on channel β-subunits. While immune tolerance may explain the lack of immunogenicity of extracellular regions, the lack of immunogenicity of most of the channel's cytoplasmic mass may be due to segment inaccessibility from organization of these regions as globular domains, to insertion of parts of these regions into the membrane phase, or to interaction with other protein elements. The definition of monoclonal antibody epitopes allows us to reinterpret previously reported monoclonal antibody competition studies, providing independent support for our model of sodium channel cytoplasmic domain structure. In addition, these data suggest additional testable hypotheses concerning the interactions of the sodium channel amino- and carboxyl-termini with each other as well as with other protein elements. Received: 4 March 1998/Revised: 15 May 1998     

Bacterial Expression,Purification and Characterization of a Rice Voltage-Dependent,Anion-Selective Channel Isoform,OsVDAC4

The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 β-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies—spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in β-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large β-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.     

Kir6.2 Channel Gating by Intracellular Protons: Subunit Stoichiometry for Ligand Binding and Channel Gating

The adenosine triphosphate-sensitive K⁺ (K_ATP) channels are gated by several metabolites, whereas the gating mechanism remains unclear. Kir6.2, a pore-forming subunit of the K_ATP channels, has all machineries for ligand binding and channel gating. In Kir6.2, His175 is the protonation site and Thr71 and Cys166 are involved in channel gating. Here, we show how individual subunits act in proton binding and channel gating by selectively disrupting functional subunits using these residues. All homomeric dimers and tetramers showed pH sensitivity similar to the monomeric channels. Concatenated construction of wild type with disrupted subunits revealed that none of these residues had a dominant-negative effect on the proton-dependent channel gating. Subunit action in proton binding was almost identical to that for channel gating involving Cys166, suggesting a one-to-one coupling from the C terminus to the M2 helix. This was significantly different from the effect of T71Y heteromultimers, suggesting distinct contributions of M1 and M2 helices to channel gating. Subunits underwent concerted rather than independent action. Two wild-type subunits appeared to act as a functional dimer in both cis and trans configurations. The understanding of K_ATP channel gating by intracellular pH has a profound impact on cellular responses to metabolic stress as a significant drop in intracellular pH is more frequently seen under a number of physiological and pathophysiological conditions than a sole decrease in intracellular ATP levels. Runping Wang, Junda Su contributed equally to this work.     

Structure,Dynamics and Implied Gating Mechanism of a Human Cyclic Nucleotide-Gated Channel

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide–binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such ‘voltage-insensitive’ channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.