Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR

The outermost lipid-exposed alpha-helix (M4) in each of the homologous alpha, beta, delta, and gamma/epsilon subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 alpha-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in alpha M4, beta M4, delta M4, and epsilon M4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select aM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 alpha-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.

Automated summary

The outermost lipid-exposed alpha-helix (M4) in each of the homologous alpha, beta, delta, and gamma/epsilon subunits of the muscle nicotinic acetylcholine receptor (nAChR) is proposed as a lipid sensor. However, the mechanism of this sensor is not clear. To explore the function of M4 alpha-helix in each subunit of the nAChR, alanine mutations were characterized in alpha M4, beta M4, delta M4, and epsilon M4. The results suggest different functional roles for M4 alpha-helix in each subunit and lipid sensing occurs through cumulative interactions at the M4-M1/M3 interface.