Effects of pipette geometry on the time course of solution change in patch clamp experiments.

The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand.     

Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses.

Completely desensitizing excitatory channels were activated in outside-out patches of crayfish muscle membrane by applying glutamate pulses with switching times of approximately 0.2 ms for concentration changes. Channels were almost completely activated with 10 mM glutamate. Maximum activation was reached within 0.4 ms with greater than or equal to 1 mM glutamate. Channel open probability decayed with a time constant of desensitization of 2 ms with 10 mM glutamate and more rapidly at lower glutamate concentrations. The rate of beginnings of bursts (average number of beginnings of bursts per time bin) decayed even faster but approximately in proportion to the glutamate concentration. The dose-response curve for the channel open probability and for the rate of bursts had a maximum double-logarithmic slope of 5.1 and 4.2, respectively. Channels desensitized completely without opening at very low or slowly rising glutamate concentrations. Desensitization thus originates from a closed channel state. Resensitization was tested by pairs of completely desensitizing glutamate pulses. Sensitivity to the second pulse returned rapidly at pulse intervals between 1 and 2 ms and was almost complete with an interval of 3 ms. Schemes of channel activation by up to five glutamate binding steps, with desensitization by glutamate binding from closed states, are discussed. At high agonist concentrations bursts are predominantly terminated by desensitization. Quantal currents are generated by pulses of greater than 1 mM glutamate, and their decay is determined by the duration of presence of glutamate and possibly by desensitization.     

Centrifugal precipitation chromatography

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.     

Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels.

In the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels convert stimulus-induced changes in the internal concentrations of cGMP and cAMP into changes in membrane potential. Although it is known that significant activation of these channels requires the binding of three or more molecules of ligand, the detailed molecular mechanism remains obscure. We have probed the structural changes that occur during channel activation by using sulfhydryl-reactive methanethiosulfonate (MTS) reagents and N-ethylmaleimide (NEM). When expressed in Xenopus oocytes, the alpha-subunit of the bovine retinal channel forms homomultimeric channels that are activated by cGMP with a K1/2 of approximately 100 microM. Cyclic AMP, on the other hand, is a very poor activator; a saturating concentration elicits only 1% of the maximum current produced by cGMP. Treatment of excised patches with MTS-ethyltrimethylamine (MTSET) or NEM dramatically potentiated the channel's response to both cyclic nucleotides. After MTSET treatment, the dose-response relation for cGMP was shifted by over two orders of magnitude to lower concentrations. The effect on channel activation by cAMP was even more striking. After modification, the channels were fully activated by cAMP with a K1/2 of approximately 60 microM. This potentiation was abolished by conversion of Cys481 to a nonreactive alanine residue. Potentiation occurred more rapidly in the presence of saturating cGMP, indicating that this region of the channel is more accessible when the channel is open. Cys481 is located in a linker region between the transmembrane and cGMP-binding domains of the channel. These results suggest that this region of the channel undergoes significant movement during the activation process and is critical for coupling ligand binding to pore opening. Potentiation, however, is not mediated by the recently reported interaction between the amino- and carboxy-terminal regions of the alpha-subunit. Deletion of the entire amino-terminal domain had little effect on potentiation by MTSET.     

Ligand Depletion in vivo Modulates the Dynamic Range and Cooperativity of Signal Transduction

Biological signal transduction commonly involves cooperative interactions in the binding of ligands to their receptors. In many cases, ligand concentrations in vivo are close to the value of the dissociation constant of their receptors, resulting in the phenomenon of ligand depletion. Using examples based on rotational bias of bacterial flagellar motors and calcium binding to mammalian calmodulin, we show that ligand depletion diminishes cooperativity and broadens the dynamic range of sensitivity to the signaling ligand. As a result, the same signal transducer responds to different ranges of signal with various degrees of cooperativity according to its effective cellular concentration. Hence, results from in vitro dose-response analyses cannot be applied directly to understand signaling in vivo. Moreover, the receptor concentration is revealed to be a key element in controlling signal transduction and we propose that its modulation constitutes a new way of controlling sensitivity to signals. In addition, through an analysis of the allosteric enzyme aspartate transcarbamylase, we demonstrate that the classical Hill coefficient is not appropriate for characterizing the change in conformational state upon ligand binding to an oligomeric protein (equivalent to a dose-response curve), because it ignores the cooperativity of the conformational change for the corresponding equivalent monomers, which are generally characterized by a Hill coefficient . Therefore, we propose a new index of cooperativity based on the comparison of the properties of oligomers and their equivalent monomers.     

Openings of the rat recombinant alpha 1 homomeric glycine receptor as a function of the number of agonist molecules bound

The functional properties of rat homomeric alpha 1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat alpha1 subunit cDNA. Whole-cell dose-response curves gave EC(50) estimates between 30 and 120 microM and a Hill slope of approximately 3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 microM) to outside-out patches. Single channel conductances were mostly 60-90 pS, but smaller conductances of approximately 40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.     

Free-energy relationships in ion channels activated by voltage and ligand

Many ion channels are modulated by multiple stimuli, which allow them to integrate a variety of cellular signals and precisely respond to physiological needs. Understanding how these different signaling pathways interact has been a challenge in part because of the complexity of underlying models. In this study, we analyzed the energetic relationships in polymodal ion channels using linkage principles. We first show that in proteins dually modulated by voltage and ligand, the net free-energy change can be obtained by measuring the charge-voltage (Q-V) relationship in zero ligand condition and the ligand binding curve at highly depolarizing membrane voltages. Next, we show that the voltage-dependent changes in ligand occupancy of the protein can be directly obtained by measuring the Q-V curves at multiple ligand concentrations. When a single reference ligand binding curve is available, this relationship allows us to reconstruct ligand binding curves at different voltages. More significantly, we establish that the shift of the Q-V curve between zero and saturating ligand concentration is a direct estimate of the interaction energy between the ligand- and voltage-dependent pathway. These free-energy relationships were tested by numerical simulations of a detailed gating model of the BK channel. Furthermore, as a proof of principle, we estimate the interaction energy between the ligand binding and voltage-dependent pathways for HCN2 channels whose ligand binding curves at various voltages are available. These emerging principles will be useful for high-throughput mutagenesis studies aimed at identifying interaction pathways between various regulatory domains in a polymodal ion channel.     

Acetylcholine-induced receptor-controlled ion flux investigated by flow quench techniques

Using a quench flow technique with membrane vesicles, the acetylcholine receptor-controlled transmembrane ion flux and the inactivation of the receptor with acetylcholine were measured in the msec time region. The ion flux was followed by influx of radioactive tracer ion and the inactivation was followed by an ion flux assay of receptor pre-incubated with ligand. The measurements covered a concentration range to complete saturation of the $\text{active}$ state of the receptor with ligand, and were consistent with a minimal model previously proposed on the basis of experiments with carbamylcholine. The ion translocation rate at saturation with acetylcholine is about twice that at saturation with carbamylcholine and this reflects a more favored channel opening equilibrium for acetylcholine.     

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.     

Single channel properties of P2X2 purinoceptors

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current-voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of approximately 30 pS at -100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was approximately 150 mM at 0 mV and voltage dependent. The binding site appeared to be approximately 0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 microM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of approximately 7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose-response curve.     

Computer simulated evolution of a network of cell-signaling molecules.

We have trained a computer model of a simple cell-signaling pathway to give specified responses to a pulse of an extracellular ligand. The pathway consists of two initially identical membrane receptors, each of which relays the concentration of the ligand to the level of phosphorylation of an intracellular molecule. Application of random "mutational" changes to the rate constants of the pathway, followed by selection in favor of certain outputs, generates a variety of wave forms and dose-response curves. The phenotypic effect of mutations and the frequency of selection both affect the efficiency with which the pathway achieves its target. When the pathway is trained to give a maximal response at a specific concentration of the stimulating ligand, it gives a consistent pattern of changes in which the two receptors diverge, producing a high-affinity form with excitatory output and a low-affinity form with inhibitory output. We suggest that some high- and low-affinity forms of receptors found in present-day cells might have originated by a similar process.     

Identification and properties of a novel type of Na+-permeable amiloride-sensitive channel in thyroid cells

Amiloride-sensitive cationic channels are present in the apical membrane of porcine thyroid cells in primary culture. An amiloride-sensitive (K0.5 = 150 +/- 28 nM where K0.5 is the concentration of unlabelled ligand which reduces the specific binding of the same labelled ligand by 50%) 22Na+-flux component (Km for Na+ at 18 mM) has been identified which was also blocked by the potent amiloride derivative phenamil (K0.5 = 47 +/- 21 nM). The most potent inhibitor of Na+/H+ exchange, ethylisopropyl-amiloride, hardly inhibited this 22Na+-influx component at a concentration of 21 microM. Amiloride binding sites were characterized using [3H]phenamil. The tritiated ligand binds to a single family of binding sites in thyroid membranes with a Kd value of 50 +/- 10 nM and a maximal binding capacity of 5 +/- 1 pmol/mg protein. Patch-clamp experiments have directly demonstrated the existence of a phenamil- and amiloride-sensitive cationic channel, with a conductance of 2.6 pS, which is permeable to sodium, but not very selective (PNa+/PK+ = 1.2). This channel is an important element in the regulation of the resting membrane potential of thyroid cells.     

Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor.

The activation kinetics of N-methyl-D-aspartate (NMDA) channels in outside-out patches from cultured hippocampal neurons were analyzed to determine the number of glutamate and glycine binding sites per channel. Following rapid steps into high concentrations of glutamate, the activation time course was concentration-independent and limited by transitions between the shut, but fully liganded state and the open state. At lower concentrations, ligand binding was rate-limiting. The resulting sigmoidal activation time course was best fitted by a kinetic model with two glutamate binding sites. Glycine concentration jumps in the continuous presence of glutamate were also best fitted with a two-site model. Agonist and co-agonist binding were better described by an independent, rather than a sequential model. We suggest that the NMDA receptor is at least a tetramer containing four ligand binding subunits, assuming a single binding site per subunit.     

Use of a receptor competition assay to explore the interaction of the T cell antigen-specific receptor with its ligands

The observation has previously been made that receptor-bearing cells in culture compete with each other for their ligand. As a result, at a fixed concentration of ligand, the fractional occupancy of the receptor will tend to fall as the number of cells is increased. We have demonstrated that T cells in culture also compete for their ligand, the combination of foreign antigen and the Ia molecule (antigen-Ia), and that this manifests itself as shifts in the antigen dose-response curves as the number of responding T cells is increased. Because of the complexity of T cell activation, modifications to the antigen that affected its stimulatory capacity (i.e., its potency) could come about by altering its interaction with either the T cell receptor or the Ia molecule. We could distinguish between these two possibilities by studying the extent to which the antigen dose-response curves shifted as the T cell number was increased. Amino acid substitutions in the antigen that affected the interaction with the T cell receptor caused changes in the dose-response curve shifts, whereas substitutions that decreased potency by other means did not cause such changes. Finally, two allelic forms of the Ia molecule that differed only slightly in their amino-terminal domain were used to present a single antigen to a T cell clone. Despite a difference in antigenic potency in the presence of these two Ia molecules, no difference was demonstrated in the avidity of the T cell receptor for either antigen-Ia combination. These results suggest that the antigen and the Ia molecule make physical contact during the process of antigen recognition, and that the potency of an antigen can vary as a result of its interaction with either the T cell receptor or the Ia molecule.     

Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: signaling through dynamic allosteric coupling

Signal transduction in neurons is a dynamic process, generally thought to be driven by transient changes in the concentration of second messengers. Here we describe a novel regulatory mechanism in which the dynamics of signaling through cyclic AMP are mediated by activity-dependent changes in the affinity of the hyperpolarization-activated, cation nonselective (HCN) channels for cAMP, rather than by changes in cAMP concentration. Due to the allosteric coupling of channel opening and ligand binding, changes in cellular electrical activity that alter the opening of the HCN channels modify the binding of static, basal levels of cAMP. These changes in ligand binding produce long-lasting changes in channel function which can contribute to the regulation of rhythmic firing patterns.     

Homology modeling of Kv1.5 channel block by cationic and electroneutral ligands

The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic “blocking particle”. A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.     

Voltage clamp analysis of inhibitory synaptic action in crayfish stretch receptor neurons

Abdominal stretch receptor neurons of Procambarus clarkii were voltage clamped with two microelectrodes, and the synaptic currents set up by stimulating the inhibitory axons, or by rapid bath application of gamma-aminobutyric acid (GABA), were recorded. The inhibitory postsynaptic current (IPSC) decay was exponential, the time constant of decay being increased by membrane depolarization. The IPSC decay was prolonged by diphenylhydantoin, whereas the IPSC amplitude was depressed by picrotoxin. It is suggested that these effects may reflect slowing of the channel closing and opening rates, respectively. Step clamps applied in the presence of GABA yield currents that show inactivation in the 100 ms time range. This inactivation was shown to reflect chloride movement across the membrane. Step clamp data were used to construct dose-response curves. Diphenylhydantoin shifts the dose-response curve to the left with little change in the maximum response. Picrotoxin shifts the curve to the right with a small reduction in the maximum response. These effects are consistent with the postulated effects on channel opening and closing rates, if GABA normally opens a large portion of the channels. Suitable combinations of picrotoxin and diphenylhydantoin acting together leave the dose-response curve unmodified, as predicted.     

The outer pore of the glutamate receptor channel has 2-fold rotational symmetry

The ligand binding domain of glutamate receptors (GluRs) has 2-fold rotational symmetry. The structure including the symmetry of the GluR ion channel remains undefined. Here we used substituted cysteines in the pore-lining M3 segment of the AMPAR GluR-A subunit and various cysteine-reactive agents to study the structure of the channel during gating. We find that cysteines substituted at A+6, located in the highly conserved SYTANLAAF motif, are grouped in pairs consistent with a 2-fold symmetry in the extracellular part of the pore. To account for this symmetry and crosslinking, we propose that the M3 segments in two neighboring GluR subunits are kinked within SYTANLAAF in opposite directions relative to the central axis of the pore. Our results extend the 2-fold rotational symmetry from the ligand binding domain to at minimum the extracellular part of the channel and suggest a model of gating movements in GluR pore-forming domains.     

Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.