Despite the open probability of the wild type KcsA being low, the single-channel current recordings obtained for the R64L, R89L and R64L/R89L double mutant clearly indicate that the replacement of the charged arginine residues at position 64 and 89 with leucine alters the gating of KcsA, reducing both its open probability and conductance. Care must be taken in the interpretation of the electrophysiology recordings as lipid protein interactions have been reported to play a significant role in the folding and stability of the tetrameric channel which would clearly have implication on the conductance behaviour [28]. However, the appearance of stable tetramers on the SDS-PAGE of the wild-type and mutants (Fig. 1) suggests that the mutant proteins are both correctly folded and assembled into stable tetramer and thus the changes observed are a result in different gating behaviour. Several patch-clamping studies have previously been reported on the R64A mutant of KcsA reconstituted by mixing bacterial membranes with asolectin vesicles [24,25]. There are significant differences in the responses of the R64A mutation between these studies with Perozo and co-workers reporting a significantly higher open probability of 65% [24] whilst Kremer et al. find that the R64A mutant has a higher tendency to close than wild type KcsA [25]. Interestingly, channel-gating behaviour was also reported to be dependent on the nature of the substitution with a R64D mutation giving rise to a higher open probability than R64A, which Kremer et al. suggested to arise from the formation of a salt bridge across the non-annular lipid binding site which may stabilise the open state thereby mimicking the presence of an anionic lipid [25]. The low open probabilities, compared to wild type KcsA, that we observe in the R64L, R89L and R64L/R89L double mutant would be consistent with such a hypothesis, suggesting that the bulky, uncharged sidechains are unable to form the necessary interactions necessary to stabilise the open state.