A comprehensive sequence analysis of the 64 full-length DmNaV clones and site-directed mutagenesis of variant-specific amino acid changes are eventually required in order to identify those specific amino acid sequences that are responsible for the gating property differences reported in this paper. Furthermore, it would be important to determine whether these specific changes are caused by RNA editing, alternative splicing, or PCR errors introduced during RT-PCR cloning. Toward this goal, we have sequenced five functional variants (DmNaV5-1 and DmNaV7-1, DmNaV1-1, DmNaV1-6 and DmNaV19) that represent a wide range of gating properties (). As mentioned above, DmNaV5-1 and DmNaV7-1 are activated at low (i.e., more hyperpolarizing) voltages, DmNaV1-6 and DmNaV19 at high (i.e., more depolarizing) voltages, and DmNaV1-1 at an intermediate voltage ( and ). Because we isolated the full-length clones by RT-PCR, we expected that some of nucleotide changes would result from random PCR errors. However, such random errors are highly unlikely if a nucleotide change occurs in independent clones that belong to different splice types. Based on this criterion, we could attribute a total of nine amino acid changes (in the upper part of ) to A-to-I RNA editing. Whether these changes are the result of RNA editing remain to be determined because some of the sequence differences could be caused by single-nucleotide polymorphism between the strain (unspecified) in the Genbank and strain W1118 used in this study.
Common and unique amino acid changes in five DmNaV variants
Four of the nine A-to-I editing () were previously reported in the DmNaV
transcript (Palladino et al., 2000
). However, these RNA editing events are detected in both low-voltage-activated and high-voltage-activated variants and are therefore not likely responsible for the unique gating properties of these variants. In addition to the A-to-I editing, E299Q and L1363F were found in all five sequenced variants; both were caused by C to G changes. Finally, at the amino acid position 260, two separate changes, one from I to V and the other from I to T, were found in DmNaV
5-1 and DmNaV
7-1, respectively. Further sequencing of the corresponding regions in other 59 full-length clones reveal one more clone, DmNaV
4-2, which belongs to a different splice type, and also contains an I260 V change. DmNaV
4-2 was not functional in Xenopus
oocytes, presumably caused by another unidentified sequence(s) in this variant. The I260 V amino acid change results from an a(tc) to g(tc) nucleotide substitution. Because it is highly unlikely that two DmNaV
cDNA clones from different splice types would have the PCR errors at the same nucleotide, we concluded that the I260 V change in DmNaV
5-1 is caused by RNA editing. Examination of the genomic sequence of the DmNaV
gene revealed the a(tc) codon at the corresponding position. Therefore, the I260 V change is the result of A-to-I editing. Next, we conducted experiments to determine whether the I260 V change in DmNaV
5-1 is responsible for the low-voltage dependence of activation. We substituted V260 with I260 in DmNaV
5-1. Interestingly, the V260I substitution shifted the voltage dependence of activation by 6 mV in the depolarizing direction (), demonstrating that this A-to-I editing event is responsible for the low-voltage dependence of activation of DmNaV
In summary, we report here the functional characterization of the largest collection of insect sodium channel variants to date. Further site-directed mutagenesis experiments are currently underway to identify potentially all amino acid changes/RNA editing events that are responsible for variant-specific gating properties. This work lays a foundation for further studies to understand the role of diverse DmNaV variants in regulating neuronal excitability in insects.