The inward rectifier K⁺channel Kir2.1 is gated by blockage by intracellular polyamines and Mg²⁺, whose occupancy of the channel is opposed by extracellular K⁺. An aspartate residue (D172) located in the second transmembrane domain (M2) and two glutamate residues (E224 and E299) in the cytoplasmic pore have been identified as binding sites for these blockers (for review, see Stanfield et al. 2002). The mechanism of channel blockage by polyamines is however not fully established. To begin to test whether polyamines come into close proximity with the channel selectivity filter, we have examined the effect of mutation of T142 in the K⁺ selectivity sequence TIGYG on channel gating. Channels were expressed in oocytes taken from Xenopus frogs. We used two-electrode voltage clamp to examine current-voltage relationships of wild type and mutant channels. Normalised conductance-voltage relationships (Fig. 1) were fitted using a Boltzmann relation with two components each of which evaluates to 0.5 at membrane potentials V₁ and V₂ with steepness factors k₁ and k₂. V₁ was altered from -44.0 ± 1.0 mV (mean ± s.e.m; n = 6) in wild type to -54.4 ± 0.5 mV (n=5) in T142A, while V₂ = -103.9 ± 8.8 (wild type) and -115.9 ± 0.7 (T142A). k₁ and k₂ were little altered: k₁ = 13.7 ± 0.3 and 12.6 ± 0.4mV in wild type and T142A, respectively; k₂ = 20.6 ± 2.6 and 24.1 ± 0.8mV. The shift in V₁ to more negative values in the T142A mutant would be consistent with an increase in polyamine affinity of the mutant channels, an increase which we are in the process of testing using macropatches. Inspection of the time course of current development under hyperpolarisation is suggestive of a slower release of polyamines in the mutant. Our results are consistent with polyamines binding deeply in the channel, in part adjacent to the intracellular end of the selectivity filter. Alternatively, since the mutation may disrupt one of the K⁺ co-ordination sites in the selectivity filter (Zhou & MacKinnon, 2004), our results may also be explained by reduced competition between extracellular K⁺ and the blocking polyamine. We are currently testing these hypotheses.