Peter W. Gage

Hypoxia increases persistent sodium current in rat ventricular myocytes.

1. A persistent inward current activated by depolarization was recorded using the whole‐cell, tight seal technique in rat isolated cardiac myocytes. The amplitude of the inward current increased when cells were exposed to a solution with low oxygen tension. 2. The persistent inward current had the characteristics of the persistent Na+ current described previously in rat ventricular myocytes: it was activated at negative potentials (‐70 mV), reversed close to the equilibrium potential for Na+ (ENa), was blocked by TTX and was resistant to inactivation. 3. Persistent single Na+ channel currents activated by long (200‐400 ms) depolarizations were recorded in cell‐attached patches on isolated ventricular myocytes. Hypoxia increased the frequency of opening of the persistent Na+ channels. 4. Persistent Na+ channels recorded during hypoxia had characteristics similar to those of persistent Na+ channels recorded at normal oxygen tensions. They had a null potential at ENa, their amplitude varied with [Na+], they were resistant to inactivation and their mean open time increased with increasing depolarization. 5. The persistent Na+ channels in cell‐attached patches were blocked by TTX (50 microM) in the patch pipette and by lidocaine (100 microM). 6. It was concluded that hypoxia increases the open probability of TTX‐sensitive, inactivation‐resistant Na+ channels. The voltage dependence of these channels, and their greatly increased activity during hypoxia, suggest that they may play an important role in the generation of arrhythmias during hypoxia.

Prolongation of inhibitory postsynaptic currents by pentobarbitone, halothane and ketamine in CA1 pyramidal cells in rat hippocampus

1 Spontaneous inhibitory postsynaptic currents (i.p.s.cs) were recorded in voltage‐clamped CA1 neurones in rat hippocampal slices. 2 The exponential decay of i.p.s.cs was prolonged by concentrations of sodium pentobarbitone as low as 50 μM. With concentrations up to 100 μM, there was no change in the amplitude or rise time of the currents but current amplitude was depressed at 200 μM. The prolongation of currents increased with drug concentration within the range tested (50 to 200 μM). 3 Halothane, at concentrations from 1 to 5%, also increased the time constant of decay of i.p.s.cs. The effect increased with concentration and was fully reversible. 4 Ketamine, at a concentration of 0.5 mM, increased the time constant of decay of i.p.s.cs by 50 to 80% and the effect was reversible. 5 Ethanol (10–200 mM), nitrous oxide (75–80%), and caffeine (10 μM‐5 mM) had no detectable effect on the i.p.s.cs. 6 It is suggested that pentobarbitone, halothane and ketamine increase the time constant of decay of the i.p.s.cs by stabilizing the open state of channels activated by γ‐aminobutyric acid.

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.

A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents flowing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca2+ to solutions made channels formed by p7 less potassium‐ or sodium‐selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV‐1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents.

A persistent sodium current in rat ventricular myocytes.

1. The tight seal, whole‐cell, voltage‐clamp technique was used to record currents from single ventricular myocytes acutely dissociated from adult rat hearts. Subtraction of currents recorded in the presence and absence of tetrodotoxin (TTX, 50 microM) revealed a small, persistent, inward current following a much larger, transient, inward current. 2. Both currents were sodium currents because they reversed close to the sodium equilibrium potential and were depressed when choline was substituted for extracellular sodium. 3. The persistent sodium current could be recorded when the transient current had been inactivated with conditioning depolarization. Only slight inactivation of the persistent current occurred during depolarizing pulses lasting up to 900 ms. 4. A lower concentration of TTX (0.1 microM) blocked the persistent sodium current while having little effect on the transient sodium current. 5. The persistent sodium current was activated at more negative potentials than the transient sodium current. It cannot have been a window current because it was recorded at positive potentials when the transient current was completely inactivated. 6. Because the persistent and transient sodium currents had a different voltage dependence and sensitivity to TTX, it was concluded that different channels are responsible for the two currents.

Hippocampal GABA(A) channel conductance increased by diazepam

Benzodiazepines, which are widely used clinically for relief of anxiety and for sedation, are thought to enhance synaptic inhibition in the central nervous system by increasing the open probability of chloride channels activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Here we show that the benzodiazepine diazepam can also increase the conductance of GABAAchannels activated by low concentrations of GABA (0.5 or 5 μM) in rat cultured hippocampal neurons. Before exposure to diazepam, chloride channels activated by GABA had conductances of 8 to 53 pS. Diazepam caused a concentration-dependent and reversible increase in the conductance of these channels towards a maximum conductance of 70–80 pS and the effect was as great as 7-fold in channels of lowest initial conductance. Increasing the conductance of GABAAchannels tonically activated by low ambient concentrations of GABA in the extracellular environment may be an important way in which these drugs depress excitation in the central nervous system. That any drug has such a large effect on single channel conductance has not been reported previously and has implications for models of channel structure and conductance.

A large anion-selective channel has seven conductance levels

Ion channels have generally been found to have two predominant conductance levels thought to be associated with ‘open’ and ‘closed’ states, but intermediate (subconductance) states have also been reported1–3. We have now found that a large conductance, anion-selective channel in pulmonary alveolar epithelial cells4 can adopt any of six open levels of conductance that are integer multiples of 60–70 pS. The channel is usually either fully open or fully closed. The frequencies of the different conductance levels are inconsistent with the notion that there are six independent channels. We suggest that the channel consists of six conducting pathways in parallel, ‘co-channels’, with a shared gating mechanism that can synchronously render all of them non-conducting. Other channels with lower maximum conductance may operate in a similar way but multiple conductance levels would not easily be detected because of a less favourable signal-to-noise ratio.

SARS coronavirus E protein forms cation-selective ion channels.

Abstract Severe Acute Respiratory Syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). Coronaviruses including SARS-CoV encode an envelope (E) protein, a small, hydrophobic membrane protein. We report that, in planar lipid bilayers, synthetic peptides corresponding to the SARS-CoV E protein forms ion channels that are more permeable to monovalent cations than to monovalent anions. Affinity-purified polyclonal antibodies recognizing the N-terminal 19 residues of SARS-CoV E protein were used to establish the specificity of channel formation by inhibiting the ion currents generated in the presence of the E protein peptides.

INHIBITION OF OXIDATIVE METABOLISM INCREASES PERSISTENT SODIUM CURRENT IN RAT CA1 HIPPOCAMPAL NEURONS

1 Whole‐cell patch‐clamp recordings from freshly dissociated rat CA1 neurons revealed a large transient Na+ current (INa,T) and a smaller, inactivation‐resistant persistent Na+ current (INa,P). Both currents could be blocked with TTX. 2 The average current densities of INa,T and INa,P in thirty cells were 111.0 ± 9.62 and 0.87 ± 0.13 pA pF−1, respectively. 3 Inhibiting oxidative phosphorylation by adding 5 mM sodium cyanide to the pipette solution significantly increased the amplitude of INa,P but had no significant effect on the amplitude of INa,T. 4 Exposing CA1 neurons to hypoxia for more than 7 min caused an increase in the amplitude of INa,P. There was also a delayed decrease in the amplitude of INa,T. 5 I Na,P was more sensitive to the Na+ channel blockers TTX and lidocaine than INa,T. The IC50 for the effect of TTX on INa,P was 9.1 ± 1.2 nM whereas the IC50 for INa,T was 37.1 ± 1.2 nM, approximately 4‐fold higher. Lidocaine (lignocaine; 1 μM) reduced INa,P to 0.24 ± 0.15 of control (n= 4) whereas INa,T was essentially unaffected (0.99 ± 0.11, n= 4). 6 These results show that INa,P is increased when oxidative metabolism is blocked in CA1 neurons. The persistent influx of Na+ through non‐inactivating Na+ channels can be blocked by concentrations of Na+ channel blockers that do not affect INa,T.

Nitric oxide increases persistent sodium current in rat hippocampal neurons

1 The effects of nitric oxide (NO) donors on whole‐cell, TTX‐sensitive sodium currents and single sodium channels in excised patches were examined in rat hippocampal neurons. The whole‐cell sodium current consisted of a large transient component (INa,t) and a smaller, inactivation‐resistant, persistent component (INa,p). 2 In acutely dissociated neurons, the amplitude of the whole‐cell INa,p increased by 60–80% within a few minutes of exposure to either of two NO donors, sodium nitroprusside (SNP, 100 μm) or S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP, 100 μm). 3 The amplitude of INa,t was not changed significantly by the same concentrations of SNP and SNAP, indicating that NO had a selective effect on INa,p. 4 Both NO donors significantly increased the mean persistent current in excised inside‐out patches from cultured hippocampal neurons. SNP at 10m100 μm increased average mean persistent current at a pipette potential (Vp) of +30 mV from −0.010 ± 0.014 pA (control) to −2.91 ± 1.41 pA (n= 10). SNAP at 3–100 μm increased the average mean inward current in six inside‐out patches from −0.07 ± 0.02 to −0.30 ± 0.08 pA (Vp=+30 mV). 5 The increase in persistent Na+ channel activity recorded in inside‐out patches in the presence of SNP or SNAP could be reversed by the reducing agent dithiothreitol (DTT, 2−5 mm) or by lidocaine (1–10 μm). 6 The average mean current recorded in the presence of SNP was 10‐fold higher than that elicited by SNAP. The time delay before an increase was observed was shorter with SNP (4.0 ± 0.8 min, n= 8) than with SNAP (8.4 ± 1.6 min, n= 7). 7 A component of the SNP molecule added on its own, 5 mm sodium cyanide (NaCN), increased mean current in excised inside‐out patches (Vp=+30 mV) from −0.06 ± 0.04 to −0.58 ± 0.21 pA (n= 19). This increase in channel activity could be blocked by 10 μm lidocaine and 2–5 mm DTT. 8 These results suggest that NO may directly increase the activity of neuronal persistent Na+ channels, but not transient Na+ channels, through an oxidizing action directly on the channel protein or on a closely associated regulatory protein in the plasma membrane.

Effects of some aliphatic alcohols on the conductance change caused by a quantum of acetylcholine at the toad end-plate.

1. The post‐synaptic effects of the aliphatic alcohols, ethanol to hexanol, were investigated at the neuromuscular junctions of toads, with particular emphasis on the effects of ethanol. 2. The alcohols increased the amplitude and duration of miniature end‐plate potentials. It is shown that this effect was due to the prolongation of the decay phase of miniature end‐plate currents (m.e.p.c.s). There was no effect of alcohols on the growth phase of m.e.p.c.s. 3. The prolonged decay of m.e.p.c.s in ethanol remained exponential and was normally sensitive to membrane potential. Prolonged m.e.p.c.s were associated with an equivalent prolongation of the mean duration of elementary events, as determined from power spectra of acetylcholine noise in 0–5 M ethanol. 4. The relationship betweeen the time constant of decay of m.e.p.c.s (tau) and the concentration of an alcohol of carbon chain length N (C‐N) was exponential, conforming to the equation tau equals tau‐s exp (B‐N‐C‐N), in which tau‐s is the decay time constant in standard solution and B‐N is a constant, different for each alcohol. 5. There was also an exponential relationship between B‐N and N, which closely followed the relationship between membrane‐buffer partition coefficient and carbon chain length for the different alcohols, indicating that the alcohols are active in the lipid phase of the post‐synaptic membrane. 6. It is suggested that the alcohols act by causing a change in the dielectric constant of the post‐synaptic membrane which forms the environment of the rate‐limiting reaction responsible for the decay of the end‐plate conductance. On the assumption that this reaction involves dipoles, it is shown that the small changes in dielectric constant, calculated from the partition coefficients of the alcohols and by assuming an initial lipid dielectric constant of 3, would give an exponential relationship between the time constant of decay of m.e.p.c.s and alcohol concentration. 7. The results support the hypothesis that the decay (but not the onset) of acetylcholine‐induced conductance changes is rate‐limited by a first‐order reaction which involves dipoles and occurs in the lipid environment of the post‐synaptic membrane.