D appears to be important for receptor function

D83 appears to be important for receptor function as a mutation to an alanine produced a non-functional channel. Interestingly, maintaining the charge as in D83E produced only a small reduction in GABA sensitivity. Other mutations at this position which included residues capable of hydrogen bonding such as D83N, D83T and D83C also produced functional channels. It had been shown in both MD simulations of the RDL receptor and a homology model of the human GABAA receptor that there is a salt bridge between residues analogous to D83 and R159 which likely play a key role in the stability of binding loops D and E (Ashby et al., 2012; Bergmann et al., 2013). R159 seems to be particular sensitive to mutations to either alanine (current study) or cytosine (Kwaka et al., 2018) which caused severely impaired receptors. Surprisingly, R159K produced non-functional receptors while the same mutation (R178K) in the Drosophila RDL receptor produced a channel that was similar in sensitivity to WT (Ashby et al., 2012). This suggests that in Hco-UNC-49, R159 may have other functions other than interacting with D83. This is also supported by the results of our MD simulations which found that while D83 and R159 interact by an ionic bond, the strength of this interaction and potential frequency was not as high as the equivalent residues in the RDL receptor. The genomes of parasitic nematodes appear to contain at least five distinct types of genes that encode various subunits of cys-loop GABA-gated chloride channel receptors (Accardi et al., 2012). Of these subunits only two, UNC-49B and LGC-38 have been shown to form functional homomeric LY 379268 disodium salt in Xenopus oocytes which is similar to the Drosophila RDL receptor (Siddiqui et al., 2010, 2012). On the other hand, subunits such as GAB-1 and LGC-37 have been shown to be unable to form functional homomeric channels but can combine to form a GABA sensitive heteromeric channel (Feng et al., 2002). However, the somewhat weak sequence similarity with classical mammalian αβγ GABA receptors makes it difficult to classify with certainty nematode subunits using mammalian nomenclature. Thus, the naming system utilized in Jones and Sattelle (2008) and Beech et al., 2010 is the most practical and useful method for classifying nematode GABA receptors. However, the knowledge that we now have gained though mutational studies, modelling and comparisons to mammalian αβγ GABA receptors can help better describe these subunits and the key functional motifs they possess. Thus, using criteria based on the presence or absence of key functional residues we suggest that GAB-1 is the only true β-like subunit in the nematode genome that provides essential GABA binding residues from loops B and C but lacks the key binding residues in the complementary loops D and E. The loop D and E residues would be provided by LGC-37 which when co-expressed with GAB-1 produces a functional heteromeric receptor (Feng et al., 2002). The LGC-37 residues indicated in Table 1 resulted in our description of LGC-37 as α-like. While both GAB-1 and LGC-37 are not direct orthologues of mammalian β or α subunits, they both appear to share several of the key residues that are essential for the function of each of these subunits. A similar rational can be used to describe the currently uncharacterized subunit LGC-36 as also α-like. This is what makes the UNC-49 receptors different compared to GABAA receptors, in that one subunit, (in this case UNC-49B) possessed all the essential requirements for GABA binding and receptor activation. However, it's important to note that the residues described here do not predict for certainty the function of uncharacterized subunits such as LGC-36 nor provide the complete story about the other nematode subunits as there are several other residues that are essential for agonist recognition and receptor function (Lynagh and Pless, 2014).
Declarations of interest