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Kcv channels from plant viruses represent the autonomous pore module of potassium channels, devoid of any regulatory domains. These small proteins show very reproducible single-channel behavior in planar lipid bilayers. Thus, they are an optimum system for the study of the biophysics of ion transport and gating. Structural models based on homology modeling have been used successfully, but experimental structural data are currently not available. Here we determine the size of the cytosolic pore entrance by studying the blocker kinetics. Blocker binding and dissociation rate constants ranging from 0.01 to 1000 ms−1 were determined for different quaternary ammonium ions. We found that the cytosolic pore entrance of KcvNTS must be at least 11 Å wide. The results further indicate that the residues controlling a cytosolic gate in one of the Kcv isoforms influence blocker binding/dissociation as well as a second gate even when the cytosolic gate is in the open state. The voltage dependence of the rate constant of blocker release is used to test, which blockers bind to the same binding site.

Amyloid β peptide (Aβ) is a key player in the development of Alzheimer’s disease (AD). It is the primary component of senile plaques in AD patients and is also found in soluble forms. Cholinergic activity mediated by α7 nicotinic receptors has been shown to be affected by Aβ soluble forms. To shed light into the molecular mechanism of this effect, we explored the direct actions of oligomeric Aβ1–40 and Aβ1–42 on human α7 by fluorescence spectroscopy and single-channel recordings. Fluorescence measurements using the conformational sensitive probe crystal violet (CrV) revealed that in the presence of Aβ α7 undergoes concentration-dependent conformational changes. Exposure of α7 to 100 pM Aβ changes CrV KD towards that of the desensitized state. However, α7 is still reactive to high carbamylcholine (Carb) concentrations. These observations are compatible with the induction of active/desensitized states as well as of a novel conformational state in the presence of both Aβ and Carb. At 100 nM Aβ, α7 adopts a resting-state-like structure which does not respond to Carb, suggesting stabilization of α7 in a blocked state. In real time, we found that Aβ is capable of eliciting α7 channel activity either in the absence or presence of the positive allosteric modulator (PAM) PNU-120596. Activation by Aβ is favored at picomolar or low nanomolar concentrations and is not detected at micromolar concentrations. At high Aβ concentrations, the mean duration of activation episodes elicited by ACh in the presence of PNU-120596 is significantly reduced, an effect compatible with slow open-channel block. We conclude that Aβ directly affects α7 function by acting as an agonist and a negative modulator. Whereas the capability of low concentrations of Aβ to activate α7 could be beneficial, the reduced α7 activity in the presence of higher Aβ concentrations or its long exposure may contribute to the cholinergic signaling deficit and may be involved in the initiation and development of AD.

Abstract: Objective: We reported an ultraslow late Na+ current (I NaL) in ventricular cardiomyocytes of human hearts. I NaL has been implicated in regulation of action potential duration in normal hearts and repolarization abnormalities in failing hearts. We have also identified sodium channel (NaCh) gating modes including bursts (BM) and late scattered openings (LSM) that together comprise I NaL; however, the contribution of these gating modes to Na+ current (I Na) remains unknown. In the present study, the late NaCh activity was recorded, analyzed, and modeled for heterologously expressed NaCh, Nav1.5, and for the native NaCh of ventricular mid-myocardial cardiomyocytes from normal and failing hearts. Methods and Results: We found that LSM gating was significantly slower in failing compared to normal myocytes and Nav1.5 (τ =474±10 vs. 299±9, and 229±12 ms, m ±SEM; P <0.05, n =5–6). Total burst length of BM decreased with depolarization and was larger in failing compared to normal myocytes and Nav1.5. A complete I Na decay was then numerically approximated as composed of NaCh populations operating in three gating modes described by separate Markov kinetic schemes: transient mode (TM), LSM, and BM. The populations of NaCh operating in each gating mode were estimated as 79.8% for TM, 20% for LSM, and 0.2% for BM, yielding an apparent four-exponential I Na decay at −30 mV (maximum I Na) (τ i ∼0.4, 4, 50, and 500 ms). Whole-cell recordings confirmed the existence of all four predicted components. The model also predicted voltage and temperature dependence of I NaL as well as I NaL increase and slower decay in failing hearts and acceleration by amiodarone. Conclusions: The early phase of Na+ current decay (<40 ms) involves all three NaCh gating modes, the intermediate phase (from 40 to 300 ms) is produced by BM+LSM, although the contribution of BM decreases with depolarization, and ultra-late decay (>300 ms) is determined solely by LSM. The concept of multi-mode composition for I NaL provides a new rationale for I NaL modulation by factors such as voltage, temperature, pharmacological agents, and pathological conditions. [Copyright &y& Elsevier]

NMDA receptors (NRs) are key signaling proteins in the central nervous system and represent important targets for drug development in several neurologic disorders. They are critically involved with fundamental brain processes, and thus indiscriminate pharmacological suppression of NR currents has seen only modest therapeutic success so far. Targeting harmful NR receptor activities while sparing the receptor’s vital functions requires a better understanding of the complexity of NR activation reaction; of the range of mechanisms that modulate discrete receptor activities; and of the consequences of this modulation on specific receptor functions. A quantitative account of the NR activation pathway was recently proposed and validated. It describes the gating reaction as a sequential, multi-step process rather than a binary, on-off switch. Alongside isoform-specific modulators, state-specific modulators may represent sophisticated interventions with high potential for narrow, functional specifi city. Here I review physiologic mechanisms that control NR responses; the salient features of the NR activation reaction; and discuss the model’s validity and its implications for drug development and characterization. [ABSTRACT FROM AUTHOR]

Purpose Methods. Sodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34–1.34 mM). Results. In symmetrical 500 mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 ± 0.6 pS, and the channel fractional open time was 0.96 ± 0.04. The activation midpoint potential was -96.2 ± 1.6 mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k1/2) of 60 nM at 0 mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC50] = 0.66 mM). The steady-state activation was shifted to more hyperpolarized potentials (-16.7 mV at 0.67 mM pentobarbital). Conclusion. In the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects. To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels. [ABSTRACT FROM AUTHOR]

Objective. – Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca2+-free experimental conditions in our previous study.Methods. – Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba2+, or a mixture of 30 mM Ca2+ and 60 mM Ba2+ to facilitate Ca2+ permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 μM) was used to reveal a putative role for calcineurin in regulation of LTCC.Results. – A mixture of Ca2+ and Ba2+ as charge carriers allowed for Ca2+ permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba2+ as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca2+ permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity.Conclusions. – We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba2+ permeation but can be compensated or overcome by a specific Ca2+-dependent stimulation with Ca2+ permeation. More complete restoration of physiological Ca2+ movements (e.g., Ca2+ release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC. [Copyright &y& Elsevier]

Objective: Prior studies of the modulation of the Na+ current by sympathetic stimulation have yielded controversial results. Separation of the Na+ and Ca2+ currents poses a problem in myocyte preparations. The gating of cloned Na+ channels is different in oocytes compared with mammalian expression systems. We have examined the sympathetic modulation of the α-subunit of the wild-type human cardiac Na+ channel (hH1) and the long QT-associated mutant, ΔKPQ, expressed in human embryonic kidney cells. Methods: Stable cell lines of hH1 and ΔKPQ were established in human embryonic kidney cells. Whole-cell and single-channel currents were measured with the patch–clamp technique. Sympathetic stimulation was effected by exposure to isoproterenol or 8-bromo-cAMP. Na+ channel activation and inactivation were determined using standard voltage clamp protocols. Ca2+: Na+ permeability ratio was determined under bi-ionic conditions. Results: We observed a qualitatively different effect of sympathetic stimulation on the cardiac Na+ current from that reported in frog oocytes: activation and inactivation kinetics were shifted to more negative potentials. This shift was similar for both hH1 and ΔKPQ. [ΔV0.5 for inactivation: 8.3±1.7 mV, p<0.001 (hH1); 6.8±0.9 mV, p<0.001 (ΔKPQ)]. Increased rate of closed-state inactivation contributed to the shifting of the inactivation–voltage relationship. Open-state inactivation was not affected as mean open times were unchanged. Reversal potential measurement in hH1 suggested a low Ca2+: Na+ permeability ratio of 0.017, uninfluenced by sympathetic stimulation. In ΔKPQ, the size of the persistent relative to the peak current was increased with 8-bromo-cAMP from 3.0±0.7% to 4.3±0.6% (p=0.056). Conclusions: Sympathetic stimulation exerts multiple effects on the gating of hH1. Similar effects are also seen in ΔKPQ which may increase arrhythmia susceptibility in long QT syndrome by modifying the Na+ channel contribution to the action potential. [ABSTRACT FROM PUBLISHER]

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline ( K=0.1 μM), indicating that they were mediated by γ-aminobutyric acid A (GABA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21±4 ms and 58±14 ms at a holding potential of −60 mV ( n=6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 μM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1±2.3 pA ( n=4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA ( n=6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system. [ABSTRACT FROM AUTHOR]

This study examines the subunit requirement for Torpedo acetylcholine receptor (AChR) channel expression and the influence of non- α-subunit deletions on single AChR-channel currents. Xenopus oocytes injected with subunit combinations deficient in single non- α-subunit mRNA transcripts display the following order of ACh sensitivity: β-less > γ-less > δ-less. Oocytes injected with only the α-subunit and one non-α-subunit display the order: αδ > αγ > αβ. These sequences indicate the effectiveness of non- α-subunit substitution is δ > γ > β. Single AChR-channel currents measured in oocytes deficient in either β or γ display conductance and voltage-sensitive burst kinetics similar to the wild-type channel. In contrast, the δ-less combination express channels with burst kinetics that are relatively faster and voltage insensitive. These results indicate that either a specific structural domain in the δ-subunit or its specific interactions with the α-subunit contribute to the voltage-dependent gating of the Torpedo AChR channel. [ABSTRACT FROM AUTHOR]

The effect of vasopressin on voltage-sensitive Ca2+ currents in the rat insulinoma cell line RINm5F has been investigated in patch-clamp whole-cell and single-channel current recording experiments. In the whole-cell recording configuration the dominant inward current in the presence of tetrodotoxin was noninactivating and had a high voltage threshold. This current was much enhanced when external Ca2+ was replaced by Ba2+ and was blocked by 1 microM nifedipine. It can therefore be classified as an L-current. Vasopressin enhanced the L-current without changing the voltage threshold of activation or the voltage at which the peak current was observed. Vasopressin effects were seen at concentrations as low as 0.01 nM, and the maximal effect was observed at about 1 nM. In higher concentrations the vasopressin effects were weaker, with effects at 50 nM of about the same magnitude as at 0.01 nM. In single-channel current recording experiments carried out with the cell-attached configuration there were no effects on single L-channel currents when vasopressin was added to the bath solution, but in experiments in which vasopressin (5 nM) was infused into the patch pipette a marked increase in the apparent channel open state probability was observed. We conclude that vasopressin, a peptide that is known to markedly enhance glucose-evoked insulin secretion, stimulates opening of the voltage-sensitive Ca2+ channels in insulin-secreting cells. [ABSTRACT FROM AUTHOR]

Extensive chemical kinetic measurements of acetylcholine receptor-controlled ion translocation in membrane vesicles isolated from the electroplax of Electrophorus electricus have led to the proposal of a minimum model which accounts for the activation, desensitization, and voltage-dependent inhibition of the receptor by acetylcholine, suberyldicholine, and carbamoylcholine. Comparison of chemical kinetic measurements of the dynamic properties of the acetylcholine receptor in vesicles with the properties of the receptor in cells obtained from the same organ and animal have been hampered by an inability to make the appropriate measurements with Electrophorus electricus electroplax cells. Here we report a method for exposing and cleaning the surface of electroplax cells obtained from both the Main electric organ and the organ of Sachs and the results of single-channel current recordings which have now become possible. The single-channel current recordings were made in the presence of either carbamoylcholine or suberyldicholine, as a function of temperature and transmembrane voltage. Both the channel open times and the single-channel conductance were measured. The data were found to be consistent with the model based on chemical kinetic measurements using receptor-rich membrane vesicles prepared from the Main electric organ of E. electricus. [ABSTRACT FROM AUTHOR]