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. 2014 May 21;34(21):7238–7252. doi: 10.1523/JNEUROSCI.5105-13.2014

Figure 9.

Figure 9.

Effects of M3–M4 linker mutations on the kinetic properties of the α1 GlyR. A, Sequence alignment of the wild-type and M3–M4 linker mutant α1 GlyRs under study. The “//” in the wild-type α1 GlyR and H311S sequences indicates a break in the sequence meant only to increase the clarity of the alignment. B, Time courses of deactivation. Recordings from the GlyRM3M4 311H construct were most often best fit with a single exponential component, unlike recordings from all other constructs discussed here (Table 4); the representative trace decays to a zero-current level faster than those of all other constructs because it does not exhibit the slow component of decay. C, Time courses of entry into desensitization. Of all the tested constructs, the GlyRM3M4 311H desensitized the fastest. Addition of the K24′A mutation (which, when engineered on the wild-type background, slowed down desensitization to a large degree; Fig. 7F) to this construct slowed down desensitization as well, but the time course of the GlyRM3M4 311H + K24′A channel remained faster than that of the K24′A single-point mutant. Thus, we conclude that the large deletion of the M3–M4 linker engineered in the GlyRM3M4 311H construct has a comparatively large effect on entry into desensitization. The GlyRM3M4 8xAla construct, on the other hand, desensitized the slowest. D, Time courses of recovery from desensitization. Data were gathered and analyzed using the same methods as for Figure 4. Most constructs recovered at a rate similar to that of the wild-type α1 GlyR (Table 4). The data points corresponding to the GlyRM3M4 311S, GlyRM3M4 311A, and GlyRM3M4 311H constructs and H311S point mutant nearly “fall” on top of each other, making them difficult to distinguish. The GlyRM3M4 8xAla exhibited markedly faster recovery than the wild-type (Table 4). B, C, Dashed line indicates the current baseline. B–D, The concentration of Gly during the ligand pulses was 10 mm, and the membrane potential was held constant at −80 mV. All recordings were performed using the outside-out configuration.