Unc-1: a stomatin homologue controls sensitivity to volatile anesthetics in Caenorhabditis elegans

Abstract

To identify sites of action of volatile anesthetics, we are studying genes in a functional pathway that controls sensitivity to volatile anesthetics in the nematode Caenorhabditis elegans. The unc-1 gene occupies a central position in this pathway. Different alleles of unc-1 have unique effects on sensitivity to the different volatile anesthetics. UNC-1 shows extensive homology to human stomatin, an integral membrane protein thought to regulate an associated ion channel. We postulate that UNC-1 has a direct effect on anesthetic sensitivity in C. elegans and may represent a molecular target for volatile anesthetics.

Figure 1

Histogram showing changes in EC₅₀ values of unc-79, unc-1, and unc-79;unc-1 from those of N2 (baseline) in four anesthetics. H, halothane; E, enflurane; ISO, isoflurane; DE, diethyl ether. Note the ability of unc-1 to suppress the increased sensitivities of unc-79 to halothane. unc-1 also suppresses the decreased sensitivities of unc-79 to enflurane. unc-1 by itself has an increased sensitivity to diethyl ether and does not suppress the increased sensitivity of unc-79 to ether. The null allele of unc-1 used in these studies is e719, which behaves like e580 in other comparative tests of phenotype and anesthetic responses. EC₅₀ values, standard errors, and comparisons among strains were calculated as described by Waud (23). For each anesthetic, the EC₅₀ values of all strains were compared by using an analysis of variance (24). Significance was defined as P < 0.01. Those EC₅₀ values significantly different than the EC₅₀ value of N2 are noted with an asterisk (∗). The EC₅₀ values for halothane in the experiments were 3.2% for N2, 0.7% for unc-79, 3.5% for unc-1, and 3.3% for unc-79;unc-1. These values were used as a guide for the concentrations used to test for rescue of unc-1 described in Results.

Figure 2

Restriction map of the K03E6 cosmid (0–14 kb) showing the positions of the K03E6.5 (stomatin-like; solid boxes) and K03E6.3 (neurocalcin-like; open boxes) coding regions. Arrows indicate the direction of transcription of each gene. The thick black line shows the position of the rescuing 5.6-kb HindIII fragment. The location of the deletions in fc53 [2.3 kb (0.9–2.2 kb) in K03E6.3 and 13 bp in exon 4 of K03E6.5] are shown by the discontinuous line above the cosmid. The first two exons of K03E6.3, which lie outside the K03E6 cosmid, are shown extending beyond the thin black line representing the K03E6 cosmid.

Figure 3

Multiple alignment of the deduced amino acid sequence of UNC-1 with that of the two mammalian stomatins (15). M, mouse; H, human; *, identity; dot, similarity. Most of the unc-1 mutations (shown above the boldfaced type) lie in residues that are identical or similar between the proteins (★, stop). The two ⇓ marks indicate the region that is replaced with the following residues RTCGSEGHPSPPTTHTCHGRRSRGRA in e719. The predicted membrane-spanning domain for each protein is shaded (22). A second membrane spanning domain is also predicted for the UNC-1 protein. Cysteine residues within the hydrophobic stretch that may potentially be palmitoylated to provide membrane-anchoring function are underlined. The 1.2-kb K03E6.5 transcript includes a 3′ untranslated region of 343 bp in addition to an ORF of 855 bp (29).

Figure 4

Schematic representation of the way in which UNC-1 may affect membrane conductance and anesthetic sensitivity. Volatile anesthetics such as halothane (H) may bind directly to UNC-1 or to a protein that associates with UNC-1 such as the ion channel that UNC-1 regulates. Altered interactions with any of these proteins in the different unc-1 mutants would then lead to abnormal Na⁺/K⁺ fluxes in the affected cells thereby altering their normal motion and affecting their responses to volatile anesthetics. The regions changed by mutations in the 12 sequenced alleles are designated by an asterisk. Note that each asterisk may represent more than one allele; for instance, five different alleles have changes in the proposed “ball,” which is thought to plug an associated channel.