Stefan H. Heinemann

Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca2+ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.

High-quality life extension by the enzyme peptide methionine sulfoxide reductase

Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila. The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.

Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II.

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA‐derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single‐channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single‐channel conductance.

MOLECULAR DETERMINANTS FOR ACTIVATION AND INACTIVATION OF HERG, A HUMAN INWARD RECTIFIER POTASSIUM CHANNEL

1. The human eag‐related potassium channel, HERG, gives rise to inwardly rectifying K+ currents when expressed in Xenopus oocytes. 2. The apparent inward rectification is caused by rapid inactivation. In extracellular Cs+ solutions, large outward currents can be recorded having an inactivation time constant at 0 mV of about 50 ms with an e‐fold change every 37 mV. 3. HERG channel inactivation is not caused by an amino‐terminal ball structure, as a deletion of the cytoplasmic amino terminus (HERG delta 2‐373) did not eliminate inactivation. However, channel deactivation was accelerated about 12‐fold at ‐80 mV. 4. Mutation of S631 to A, the homologous residue of eag channels, in the outer mouth of the HERG pore completely abolished channel inactivation. 5. Activity of HERG channels depended on extracellular cations, which are effective for channel activation, in the order Cs+ > K+ > > Li+ > Na+. The point mutation S631A strongly reduced this channel regulation. 6. By analogy to functional aspects of cloned voltage‐gated potassium channels, rectification of HERG, as well as its kinetic properties during the course of an action potential, are presumably governed by a mechanism reminiscent of C‐type inactivation.

Regulation of cell function by methionine oxidation and reduction

Reactive oxygen species (ROS) are generated during normal cellular activity and may exist in excess in some pathophysiological conditions, such as inflammation or reperfusion injury. These molecules oxidize a variety of cellular constituents, but sulfur‐containing amino acid residues are especially susceptible. While reversible cysteine oxidation and reduction is part of well‐established signalling systems, the oxidation and the enzymatically catalysed reduction of methionine is just emerging as a novel molecular mechanism for cellular regulation. Here we discuss how the oxidation of methionine to methionine sulfoxide in signalling proteins such as ion channels affects the function of these target proteins. Methionine sulfoxide reductase, which reduces methionine sulfoxide to methionine in a thioredoxin‐dependent manner, is therefore not only an enzyme important for the repair of age‐ or degenerative disease‐related protein modifications. It is also a potential missing link in the post‐translational modification cycle involved in the specific oxidation and reduction of methionine residues in cellular signalling proteins, which may give rise to activity‐dependent plastic changes in cellular excitability.

Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels.

Haem is essential for living organisms, functioning as a crucial element in the redox-sensitive reaction centre in haemproteins. During the biogenesis of these proteins, the haem cofactor is typically incorporated enzymatically into the haem pockets of the apo-haemprotein as the functionally indispensable prosthetic group. A class of ion channel, the large-conductance calcium-dependent Slo1 BK channels, possesses a conserved haem-binding sequence motif. Here we present electrophysiological and structural evidence showing that haem directly regulates cloned human Slo1 channels and wild-type BK channels in rat brain. Both oxidized and reduced haem binds to the hSlo1 channel protein and profoundly inhibits transmembrane K+ currents by decreasing the frequency of channel opening. This direct regulation of the BK channel identifies a previously unknown role of haem as an acute signalling molecule.

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes

The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K+ channels, including the human ether‐a‐go‐go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and IsK, the A‐type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μm haloperidol). In contrast, haloperidol blocked HERG channels potently with an IC50 value of approximately 1 μm. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC50 value of 2.6 μm. Haloperidol block was use‐ and voltage‐dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested. The channel mutant HERG S631A has been shown to exhibit greatly reduced C‐type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild‐type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

Functional characterization of Kv channel beta-subunits from rat brain.

1. The potassium channel beta‐subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1‐110. 2. Kv beta 1.1 was co‐expressed in Xenopus oocytes with various other potassium channel alpha‐subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2‐28 and Kv4.1 channels could be observed. 3. The second member of the beta‐subunit subfamily, Kv beta 2, had a shorter N‐terminal end, accelerated inactivation of the A‐type channel Kv 1.4, but did not induce inactivation when co‐expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co‐assembles with Kv alpha‐subunits, the N‐terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N‐terminus of Kv beta 2. The chimaeric beta‐subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha‐subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2‐28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about ‐10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.

Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing.

Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke and diabetes. The underlying mechanisms of these disorders, which are intimately associated with inflammation and oxidative stress caused by excess reactive oxygen species (ROS), have remained elusive. Here we report that ROS are powerful inhibitors of vascular smooth muscle calcium-dependent Slo1 BK or Maxi-K potassium channels, an important physiological determinant of vascular tone. By targeting a cysteine residue near the Ca2+ bowl of the BK α subunit, H2O2 virtually eliminates physiological activation of the channel, with an inhibitory potency comparable to a knockout of the auxiliary subunit BK β1. These results reveal a molecular structural basis for the vascular dysfunction involving oxidative stress and provide a solid rationale for a potential use of BK openers in the prevention and treatment of cardiovascular disorders.

NONSTATIONARY NOISE-ANALYSIS AND APPLICATION TO PATCH CLAMP RECORDINGS

Publisher Summary This chapter discusses studies of nonstationary fluctuations of sodium currents in bovine adrenal chromaffin cells for estimating the temperature and pressure dependence of the conductance of voltage-activated sodium channels as an application of the analysis method. The procedure presented allows a rapid analysis of noise records obtained under nonideal experimental conditions based on objective selection criteria. Although single-channel analysis surpasses noise analysis in many instances, there are still regimes, where it cannot be successfully applied for various reasons. In this regard, nonstationary noise analysis retain its value for electrophysiological research in particular, as ever fainter electrical signals are being investigated in biological membranes. To demonstrate the methods, the temperature and pressure dependence of the sodium channel conductance are measured, and in both respects, the sodium channel shows features similar to other ion channels. Both findings are in accord with the physical picture of a rather free ion diffusion through the channel pore which, unlike the channel gating mechanism, does not involve protein rearrangements associated with measurable activation volumes.