PPK and RPK are novel Drosophila
members of the DEG/ENaC protein superfamily (39
). The two proteins share several features. First, their primary sequence is similar and they appear to represent a new subfamily. A feature that differentiates PPK and RPK from other family members is a unique domain in the large extracellular region (Fig. B
). In PPK this domain is mostly encoded by one exon, perhaps suggesting its evolutionary origin. Much of the variability, as well as most of the conservation, between different DEG/ENaC proteins and DEG/ENaC protein subfamilies occurs in the extracellular region. The location of inserts within the extracellular region allows a grouping of the subfamilies (Fig. B
). However, we do not know the functional significance of the extracellular region. This region may form a ligand-binding site in FaNaCh and BNaC2 (32
). In addition, genetic studies with C. elegans
DEG/ENaC proteins suggest an interaction with proteins in the extracellular matrix and perhaps a role in channel gating (13
). Thus, it is possible that the unique extracellular sequences in PPK and RPK confer unique interactions and possibly regulation on these channels.
A second similarity is that both PPK and RPK appear to be ion channel subunits. The data is most direct for RPK which produced channels when expressed in Xenopus oocytes. In this regard, RPK is similar to αENaC, δNaCh, and BNCl, which generate small amiloride-sensitive Na+ currents when expressed alone. Several observations suggest that PPK is also a channel subunit: the sequence of PPK is similar to that of RPK; PPK associated with the related channel BNC1 as assessed by coimmunoprecipitation; and when coexpressed, PPK reduced current generated by BNC1.
Despite their similarities, PPK and RPK have significant differences. For example, the DEG mutation activated RPK, but not PPK (not shown). Another striking difference was that PPK, but not RPK, was expressed in the PNS of embryos and larvae. In addition, RPK transcripts were probably maternally derived, whereas PPK was expressed late in embryogenesis and in larvae. These data, plus the finding that coexpressing PPK with RPK did not alter RPK currents, indicate that the two subunits are not part of the same channel complex.
The expression pattern and function of RPK suggest it is a Na+ channel involved in early development. Amiloride-sensitive Na+ channels in mammalian embryos play an important role in fluid transport across the trophectoderm and in formation of the blastocyst (2). Like RPK, these Na+ channels have a low sensitivity to amiloride (Ki = 12 μM; 44).
The effect of blockers on RPK is also interesting. First, in contrast to other channels in the family, RPK is relatively insensitive to amiloride. This raises the possibility that some native DEG/ENaC channels may not be highly sensitive to amiloride. Alternatively, RPK may associate with other subunits in vivo to form channels with different amiloride sensitivity. Second, the Deg mutation increased amiloride sensitivity. An alanine at position 524 was dominant in conferring low sensitivity to amiloride and a valine at position 524 was dominant in activating the channel. Thus, the residue at the position of the Deg mutation influenced both channel activation and amiloride sensitivity. Third, although all DEG/ENaC proteins known to function as channels are blocked by amiloride, RPK is the first DEG/ENaC channel shown to be blocked by gadolinium. Interestingly, the doses of gadolinium required for inhibition of RPKA524V
were similar to those needed for block of mechanosensitive channels in rat supraoptic neurons (41
). Nevertheless, both amiloride and gadolinium can have targets other than DEG/ENaC proteins and stretch-activated channels. Thus, sensitivity to these agents by itself does not necessarily imply a role for RPK in mechanosensation.
PPK was expressed in a subset of the da type of md neurons and, to our knowledge, is the first described protein expressed exclusively in md neurons. da neurons are distinguished by their dendritic network that extends beyond segmental boundaries, arborizes extensively, and ramifies beneath the epidermis (3
). The dendrites from one da neuron often overlap considerably with dendrites from other da neurons, and the full complement of da dendrites gives the appearance of a “spiderweb” that covers the embryo. PPK expression was detectable only late in da neuron differentiation, suggesting that PPK is not involved in neuronal development, but may play a role in sensory function.
md neurons are found in most, if not all, insect species, and the structure and function of md neurons have been extensively characterized in several species (14
). In adult insects, md neurons are touch or stretch receptors that monitor a wide range of mechanical stimuli, including muscle tension, gut motility, and limb and wing position. Interestingly, most mechanosensation in humans occurs via free nerve endings that are morphologically similar to insect md neurons (1
). In several ways, the da neurons of Drosophila
embryos and larvae closely resemble the larval subepidermal da neurons of the blowfly, Phormia regina
. In Phormia
, each larval abdominal hemisegment possesses 15 da neurons that are located directly beneath the epidermal cell layer and send out a meshwork of dendrites that terminate on the epidermis (14
). Ultrastructural studies of these dendrites show that they penetrate the basement membrane and directly contact the basolateral surface of epidermal cells. Furthermore, since mechanical stimuli increase the frequency of neuronal firing, the subepidermal da neurons of Phormia
larvae are clearly mechanoreceptive, and are positioned to sense both touch and internal mechanical forces (14
). Thus, the cellular location of PPK suggests a mechanosensory function. Along the dendrites of PPK-stained da neurons, PPK was observed in varicosities. In stretch-sensitive da neurons of the butterfly, Pieris rapae crucivora
, similar dendritic varicosities are sites of epithelial contact and are thought to be sites of mechanotransduction (48
). Of note, Kernan et al. (29
) have produced Drosophila
mutants with mechanosensory defects. However, the neurons affected by those mutations innervate bristles and thus are probably not da neurons and do not express PPK.
Might PPK be a mechanosensor? Our evidence suggesting that PPK may be an ion channel subunit is consistent with theoretical and experimental data that mechanosensors are ion channels (25
). Although studies with blockers are not definitive, the notion is consistent with the ability of amiloride and gadolinium to block the related RPK channel at doses similar to those needed to block mechanosensation in vivo (19
). Finally, its restricted localization to a subset of da neurons suggests that PPK is in the appropriate place to play an important role in mechanosensation.