Contribution of LEV-8 subunit to the kinetics of activation and desensitization of C. elegans muscle levamisole-sensitive nicotinic receptors

GUILLERMINA HERNANDO; DIEGO RAYES; CECILIA BOUZAT

San Diego, CA. U.S.A

Congreso; 40th annual meeting of Society for Neuroscience; 2010

Society for Neuroscience. SFN.

Nicotinic acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels that mediate fast synaptic transmission. In nematodes, the muscle levamisole-sensitive AChR (L-AChR) is a target of anthelmintic drugs. L-AChR from C. elegans muscle appears to be composed of three essential (UNC-63, UNC-38, and UNC-29) and two accessory subunits (LEV-1 and LEV-8). We explored the contribution of alpha-type LEV-8 subunit to the kinetics of activation and desensitization of L-AChRs from C. elegans muscle cultured cells. Single-channel activity of L-AChRs can be readily detected from muscle cells derived from LEV-8 null mutant strain (lev-8(x-15)), thus confirming that LEV-8 is not an essential subunit. Channel conductance is similar to that of wild-type L-AChRs (~36 pS). In contrast, the duration of the slowest open component differs from that of wild-type L-AChRs, being about 2.5-fold more prolonged (0.32±0.04 and 0.70±0.11 ms for wild-type and mutant, respectively). A dramatic difference between recordings from LEV-8 null mutant and wild-type muscle cells is a time-dependent reduction in the frequency of opening events. Thus, whereas channel activity remains constant during the course of the recording in wild-type cells, it disappears within the first three minutes in the null mutant. Such a reduction is compatible with enhanced desensitization. To investigate this, we recorded macroscopic currents elicited by rapid application of levamisole or ACh in the whole-cell configuration. Our results show that the mean amplitude of currents from the null mutant is similar to that from wild-type strain. However, the decay time constant is ~4-fold reduced in the mutant, indicating faster desensitization. In addition, the steady state current, which represents receptors that remain active during the agonist-pulse, is smaller in the null mutant (41±10% and 19±10% of the peak current for wild-type and mutant, respectively). Taken together, our results reveal that L-AChRs lacking LEV-8 show increased open channel lifetime and enhanced desensitization. Thus, the properties of L-AChRs-mediated responses in muscle cells can be substantially changed whether or not accessory LEV-8 subunit is incorporated into the pentameric receptor.