Structural elements near the C-terminus are responsible for changes in nicotinic receptor gating kinetics following patch excision.

We have studied the effect of patch excision on the gating kinetics of muscle nicotinic acetylcholine receptors transiently expressed in HEK 293 cells. The experiments were performed on embryonic and adult wild-type, and several mutated, receptors using acetylcholine, carbamylcholine and tetramethylammonium as agonists. We show that patch excision of cell-attached patches into the inside-out configuration led to a reduction of mean open duration in receptors containing a γ-subunit (embryonic) but not an ɛ-subunit (adult receptors). Kinetic analysis of an embryonic receptor containing a mutated residue, αY93F, showed that the reduction in the mean open duration upon patch excision was mainly caused by an increase in the channel closing rate constant. This was confirmed by experiments on embryonic wild-type receptors using carbamylcholine as an agonist with low efficacy. By expressing receptors containing chimeric γ-ɛ subunits we found that segments of the γ-subunit corresponding to a region within the M3-M4 linker (the amphipathic helix, HA) and the M4 transmembrane domain were required for the reduction in channel open duration after excision. The results indicate that particular residues in both M4 and HA are required to allow the change in open time after excision. This finding suggests that there is an interaction between these two regions in determining the modulation of gating kinetics. The nicotinic receptor found at the skeletal muscle neuromuscular junction is among the best studied of the ligand-gated ion channels. The receptor is a pentamer. It occurs in two forms: embryonic receptors are composed of two α-subunits, and one each of β-, δ- and γ-subunits; in the adult receptor, the γ-subunit is replaced by a homologous ɛ-subunit. The activation kinetics of the two types of receptors have been extensively studied (Sine et al. 1990; Auerbach, 1993; Aylwin & White, 1994; Zhang et al. 1995; Akk & Auerbach, 1996; Maconochie & Steinbach, 1998), and a major difference between the two has been shown to occur in the channel closing rate. This rate is about 4-fold larger in the adult form. The rates for steps in the activation of both types of muscle nicotinic receptors depend on the structure of the subunits, and can be altered by mutations in several regions (Sine et al. 1995; Wang et al. 1997; Akk et al. 1999). However, activation steps do not seem to be affected by post-translational manipulations such as phosphorylation. One exception is the report that the mean open time for embryonic receptors changes after excision of the patch (Trautmann & Siegelbaum, 1983; Covarrubias & Steinbach, 1990). The mean apparent open time is reduced by 2- to 3-fold following excision, over a period of about 10 min (Covarrubias & Steinbach, 1990). It was concluded that an increase in the channel closing rate was the major reason for the change, although possible changes in the channel opening rate or acetylcholine dissociation rate were not ruled out as less important contributors (Covarrubias & Steinbach, 1990). We are interested in this observation for two reasons. The first is that understanding the basis for the change may provide some insight into the mechanisms by which receptor activation is controlled. The second is that the influence of patch configuration on activation rate constants may explain some of the variability in kinetic estimates which have been made in various studies. For example, according to Aylwin & White (1994), the channel closing rate constant of mouse embryonic wild-type nicotinic acetylcholine receptor (nAChR) is 886 s−1 while Zhang et al. (1995) found it to be almost 4-fold lower (240 s−1). This difference is likely to arise from the different patch-clamp configurations used –Aylwin & White (1994) worked on outside-out patches, Zhang et al. (1995) used cell-attached patch clamp. In the present work, we have demonstrated that the influence of patch excision persists when embryonic receptors are expressed in non-muscle-derived cells. We have used both mutated subunits and low efficacy agonists to directly examine the kinetic alterations which result in the reduction of burst duration. Both approaches allow the separation of any kinetic effects into affinity- and efficacy-based components. This is so because the channel opening is slowed when the receptors are activated by carbamylcholine or tetramethylammonium (Akk & Auerbach 1996, 1999). Similarly, a mutant receptor used in the present study has a channel opening rate constant which is ∼50-fold lower compared to the wild-type receptor, making a direct determination of the channel opening rate constant technically feasible (αY93F, Auerbach et al. 1996). We used both approaches to eliminate the possibility that wild-type receptors activated by carbamylcholine (CCh) or tetramethylammonium (TMA), or mutant receptors activated by ACh, behave qualitatively differently from ACh-activated wild-type receptors. Both approaches confirmed that an increase in the channel closing rate is the major underlying cause for excision-elicited shortening of burst durations. We have extended the observations by demonstrating that there is no apparent effect of excision on adult receptors. Finally, by use of chimeric subunits formed from γ- and ɛ-subunits, we have established that regions near the carboxy terminus of the subunits are required for the effect of excision. Specific residues in both the amphipathic helix (HA) region of the major cytoplasmic loop of the subunit and the M4 membrane spanning region must be present for excision to have an effect. It is interesting that these regions are the same as those shown earlier (Bouzat et al. 1994) to underlie the characteristic difference in burst durations seen for adult and embryonic receptors activated by acetylcholine.