The dominant mutation
exp-3(
n2372) results in worms defective in egg-laying and enteric muscle contraction. We have mapped
exp-3(
n2372) to a gene encoding a putative small conductance calcium-activated K+ (KCNN) channel (B0399.1). The phenotypes can be suppressed by feeding worms B0399.1 RNAi bacteria. Sequencing confirmed a missence mutation in the channel in the linker between transmembrane regions S4 and S5. In an effort to find loss of function alleles and interacting channel subunits we conducted a suppressor screen on
exp-3(
n2372) We screened approximately 90.000 haploid genomes and isolated 32 suppressors, all of which were intragenic revertants. Many revertants are early stop codons or mis-sense mutations affecting regions important for channel ion selectivity, function or folding. Two putative
exp-3 null alleles are moderately defective for enteric muscle contraction. One class of revertants affect the S4-S5 linker region close to the
exp-3 mutation and may not be loss of function alleles. A transcriptional GFP fusion to the short c-isoform of B0399.1 shows expression in the vulval muscles, anal depressor muscle as well as a few neurons. The expression pattern and phenotypes are consistent with a direct role for EXP-3 in modulating the excitability of the anal depressor muscles. We hypothesize that
exp-3 results in an inappropriately activated K+ channel leading to loss of muscle excitability. We interpret the results of the suppressor screen as evidence that no non-essential subunits are necessary for EXP-3 localization or function. Sequence comparison between EXP-3 and mammalian homologs indicate limited overall homology but high identity for residues mutated in the suppressor screen. We are interested in learning how the
exp-3 mutation affects channel function and if the same residue is important for mammalian KCNN channel gating. In an effort to study this we have mutated the equivalent residue in mammalian homologs of EXP-3 and will express these in mammalian cells to determine the effect on gating and currents.