Rainer Waldmann

Molecular cloning of a non-inactivating proton-gated Na^+ channel specific for sensory neurons

We have cloned and expressed a novel proton-gated Na+ channel subunit that is specific for sensory neurons. In COS cells, it forms a Na+ channel that responds to a drop of the extracellular pH with both a rapidly inactivating and a sustained Na+ current. This biphasic kinetic closely resembles that of the H+-gated current described in sensory neurons of dorsal root ganglia (1). Both the abundance of this novel H+-gated Na+ channel subunit in sensory neurons and the kinetics of the channel suggest that it is part of the channel complex responsible for the sustained H+-activated cation current in sensory neurons that is thought to be important for the prolonged perception of pain that accompanies tissue acidosis (1, 2).

H(+)-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels.

Novel members of the amiloride-sensitive Na+ channel/ degenerin family of ion channels were discovered recently. With the cloning of four mammalian H(+)-gated cation channel subunits, the first members of a novel class of ligand-gated cation channels were identified. H(+)-gated cation channel subunits are expressed in the central and peripheral nervous system. In sensory neurones, they are thought to be involved in the perception of pain that accompanies tissue acidosis.

Isolation of a Tarantula Toxin Specific for a Class of Proton-gated Na+ Channels

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H+-gated Na+ channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC50 = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

Expression cloning of an epithelial amiloride-sensitive Na+ channel: A new channel type with homologies to Caenorhabditis elegans degenerins

A complementary DNA encoding an amiloride‐sensitive Na+ channel has been cloned and characterized from rat colon. The protein encoded by the cDNA has a sequence of 699 amino acids (79 kDa) containing several putative membrane spanning domains and potential phosphorylation sites. It forms a channel that has the electrophysiological and pharmacological properties characteristic of the epithelial Na+ channel. Homologies (including in transmembrane domains) have been found between a part of the channel sequence and the Mec4 gene product of Caenorhabditis elegans, a protein associated with mutation‐induced neuronal degeneration.

The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties.

Proton-gated cation channels are acid sensors that are present in both sensory neurons and in neurons of the central nervous system. One of these acid-sensing ion channels (ASIC) has been recently cloned. This paper shows that ASIC and the mammalian degenerin MDEG, which are colocalized in the same brain regions, can directly associate with each other. Immunoprecipitation of MDEG causes coprecipitation of ASIC. Moreover, coexpression of ASIC and MDEG subunits in Xenopus oocytes generates an amiloride-sensitive H+-gated Na+ channel with novel properties (different kinetics, ionic selectivity, and pH sensitivity). In addition, coexpression of MDEG with mutants of the ASIC subunit can create constitutively active channels that become completely nonselective for Na+ versusK+ and H+-gated channels that have a drastically altered pH sensitivity compared with MDEG. These data clearly show that ASIC and MDEG can form heteromultimeric assemblies with novel properties. Heteromultimeric assembly is probably used for creating a diversity of H+-gated cation channels acting as neuronal acid sensors in different pH ranges.

H+‐Gated Cation Channelsa

ABSTRACT: H+‐gated cation channels are members of a new family of ionic channels, which includes the epithelial Na+ channel and the FMRFamide‐activated Na+ channel. ASIC, the first member of the H+‐gated Na+ channel subfamily, is expressed in brain and dorsal root ganglion cells (DRGs). It is activated by pHe variations below pH 7. The presence of this channel throughout the brain suggests that the H+ might play an essential role as a neurotransmitter or neuromodulator. The ASIC channel is also present in dorsal root ganglion cells, as is its homolog DRASIC, which is specifically present in DRGs and absent in the brain. Since external acidification is a major factor in pain associated with inflammation, hematomas, cardiac or muscle ischemia, or cancer, these two channel proteins are potentially central players in pain perception. ASIC activates and inactivates rapidly, while DRASIC has both a fast and sustained component. Other members of this family such as MDEG1 and MDEG2 are either H+‐gated Na+ channels by themselves (MDEG1) or modulators of H+‐gated channels formed by ASIC and DRASIC. MDEG1 is of particular interest because the same mutations that produce selective neurodegeneration in C. elegans mechanosensitive neurons, when introduced in MDEG1, also produce neurodegeneration. MDEG2 is selectively expressed in DRGs, where it assembles with DRASIC to radically change its biophysical properties, making it similar to the native H+‐gated channel, which is presently the best candidate for pain perception.

ASIC‐like, proton‐activated currents in rat hippocampal neurons

The expression of mRNA for acid sensing ion channels (ASIC) subunits ASIC1a, ASIC2a and ASIC2b has been reported in hippocampal neurons, but the presence of functional hippocampal ASIC channels was never assessed. We report here the first characterization of ASIC‐like currents in rat hippocampal neurons in primary culture. An extracellular pH drop induces a transient Na+ current followed by a sustained non‐selective cation current. This current is highly sensitive to pH with an activation threshold around pH 6.9 and a pH0.5 of 6.2. About half of the total peak current is inhibited by the spider toxin PcTX1, which is specific for homomeric ASIC1a channels. The remaining PcTX1‐resistant ASIC‐like current is increased by 300 μm Zn2+ and, whereas not fully activated at pH 5, it shows a pH0.5 of 6.0 between pH 7.4 and 5. We have previously shown that Zn2+ is a co‐activator of ASIC2a‐containing channels. Thus, the hippocampal transient ASIC‐like current appears to be generated by a mixture of homomeric ASIC1a channels and ASIC2a‐containing channels, probably heteromeric ASIC1a+2a channels. The sustained non‐selective current suggests the involvement of ASIC2b‐containing heteromeric channels. Activation of the hippocampal ASIC‐like current by a pH drop to 6.9 or 6.6 induces a transient depolarization which itself triggers an initial action potential (AP) followed by a sustained depolarization and trains of APs. Zn2+ increases the acid sensitivity of ASIC channels, and consequently neuronal excitability. It is probably an important co‐activator of ASIC channels in the central nervous system.

Identification, functional expression and chromosomal localisation of a sustained human proton‐gated cation channel

Non‐inactivating or slowly inactivating proton‐gated cation channels are thought to play an important role in the perception of pain that accompanies tissue acidosis. We have identified a novel human proton‐gated cation channel subunit that has biphasic desensitisation kinetics with both a rapidly inactivating Na+‐selective and a sustained component. The protein shares 84% sequence identity with the proton‐gated cation channel rASIC3 (rDRASIC) from rat sensory neurones. The biphasic desensitisation kinetics and the sequence homology suggest that this novel clone (hASIC3) is the human orthologue of rASIC3 (rDRASIC). While rASIC3 (rDRASIC) requires very acidic pH (pH < 4.5) for activation of the sustained current, the non‐inactivating hASIC3 current starts to be activated when the pH decreases to below pH 6. hASIC3 is an acid sensor and might play an important role in the detection of lasting pH changes in human. We localised the hASIC3 gene to the human chromosome 7q35, 6.4 cRad telomeric from the microsatellite AFMA082XC9.

Knockout of the ASIC2 channel in mice does not impair cutaneous mechanosensation, visceral mechanonociception and hearing

Mechanosensitive cation channels are thought to be crucial for different aspects of mechanoperception, such as hearing and touch sensation. In the nematode C. elegans, the degenerins MEC‐4 and MEC‐10 are involved in mechanosensation and were proposed to form mechanosensitive cation channels. Mammalian degenerin homologues, the H+‐gated ASIC channels, are expressed in sensory neurones and are therefore interesting candidates for mammalian mechanosensors. We investigated the effect of an ASIC2 gene knockout in mice on hearing and on cutaneous mechanosensation and visceral mechanonociception. However, our data do not support a role of ASIC2 in those facets of mechanoperception.

Acid-Sensing Ion Channel 2 Is Important for Retinal Function and Protects against Light-Induced Retinal Degeneration

pH variations in the retina are thought to be involved in the fine-tuning of visual perception. We show that both photoreceptors and neurons of the mouse retina express the H+-gated cation channel subunits acid-sensing ion channel 2a (ASIC2a) and ASIC2b. Inactivation of the ASIC2 gene in mice leads to an increase in the rod electroretinogram a- and b-waves and thus to an enhanced gain of visual transduction. ASIC2 knock-out mice are also more sensitive to light-induced retinal degeneration. We suggest that ASIC2 is a negative modulator of rod phototransduction, and that functional ASIC2 channels are beneficial for the maintenance of retinal integrity.