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

Abstract

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 ligandgated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC 50 ‫؍‬ 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.Proton-gated Na ϩ -permeable channels are the simplest form of ligand-gated channels. They are present in many neuronal cell types throughout the central nervous system (1-5), suggesting an important function of these channels in signal transduction associated with local pH variations during normal neuronal activity. These channels might also play an important role in pathological situations such as brain ischemia or epilepsy, which produce significant extracellular acidification. They are also present in nociceptive neurons (1-3, 5, 6) and are thought to be responsible for the sensation of pain that accompanies tissue acidosis in muscle and cardiac ischemia (7,8), corneal injury (9), and inflammation and local infection (10, 11). It is only very recently that the first proton-gated channel, acid-sensitive ion channel (ASIC) 1 was cloned (12). The ASICs belong to a superfamily that includes amiloride-sensitive epithelial Na ϩ channels (13, 14), the FMRFamide-gated Na ϩ channel (15), and the nematode degenerins (DEGs), which probably correspond to mechano-sensitive Na ϩ -permeable channels (16). Several ASIC subunits have now been described: ASIC1a (12), ASIC1b (17), ASIC2a (18 -21), ASIC2b (22), and ASIC3 (23-25). The different subunits produce channels with different kinetics, external pH sensitivities, and tissue distribution. They can form functional homomultimers as well as heteromultimers (21,22,26). ASIC1a and ASIC1b both mediate rapidly inactivating currents following rapid and modest acidification of the external pH. However, although ASIC1a is present in both brain and afferent sensory neurons, its splice variant ASIC1b is found only in sensory neurons (17). ASIC2a forms an active H ϩ -gated channel and is abundant in the brain but essentially absent in sensory neurons, whereas its splice variant ASIC2b is present in both brain an...

Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases

“…Psalmotoxin 1 (PcTX1) is a peptide toxin which specifically inhibits the ASIC1a current [54]. It was isolated from the venom of South American tarantula Psalmopoeus Cambridge.…”

“…The toxin contains 40 amino acids cross-linked by three disulfide bridges (Figure 3). In heterologous expression systems, PcTX1 potently and specifically inhibits the acid-activated current mediated by homomeric ASIC1a subunits in a nanomolar concentration range (IC 50 < 1 nM), without affecting the currents mediated by other configurations of ASICs [54]. At concentrations that effectively inhibit the ASIC1a current, it has no effect on voltage-gated Na + , K + , Ca 2+ channels, nor an effect on several other ligand-gated ion channels tested [10].…”

“…At concentrations that effectively inhibit the ASIC1a current, it has no effect on voltage-gated Na + , K + , Ca 2+ channels, nor an effect on several other ligand-gated ion channels tested [10]. Thus, PcTX1 is so far the best known specific blocker for ASICs and an indispensable pharmacological tool for the studies of ASIC1a-mediated processes [10,[54][55][56].…”

An efficient transcriptome analysis pipeline to accelerate venom peptide discovery and characterisation

“…Toxins have also been used to probe receptor-ligand interactions at their respective molecular targets. For example, the crystal structure of ASIC1a bound to psalmotoxin-1 (Baconguis and Gouaux, 2012;Dawson et al, 2012) isolated from the spider Araneae theraphosidae (Escoubas et al, 2000) was used to map the toxin-binding domain and understand activation mechanisms of ASICs (Baconguis and Gouaux, 2012). The co-crystallisation of ASIC1a with MitTx, a pain causing Texas coral snake toxin revealed the open state conformation of the channel (Baconguis et al, 2014).…”

Ligand-gated Ion Channels

“…Further proposed roles for centrally and peripherally located ASICs are reviewed in Wemmie et al (2006) and Lingueglia (2007). The relationship of the cloned ASICs to endogenously expressed proton-gated ion channels is becoming established (Escoubas et al, 2000;Sutherland et al, 2001;Wemmie et al, 2002Wemmie et al, , 2003Wemmie et al, , 2006Diochot et al, 2004Diochot et al, , 2007Lingueglia et al, 2006;Lingueglia, 2007;Hattori et al, 2009). Heterologously expressed heteromultimers form ion channels with altered kinetics, ion selectivity, pH-sensitivity and sensitivity to blockers that resemble some of the native proton activated currents recorded from neurones (Lingueglia et al, 1997;Babinski et al, 2000, Escoubas et al, 2000, Baron et al, 2008.…”