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Biochem Biophys Res Commun
2009 Oct 23;3883:501-5. doi: 10.1016/j.bbrc.2009.08.032.
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Kir5.1 underlies long-lived subconductance levels in heteromeric Kir4.1/Kir5.1 channels from Xenopus tropicalis.
Shang L
,
Ranson SV
,
Tucker SJ
.
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The inwardly-rectifying potassium channel subunit Kir5.1 selectively co-assembles with members of the Kir4.0 subfamily to form novel pH-sensitive heteromeric channels with unique single channel properties. In this study, we have cloned orthologs of Kir4.1 and Kir5.1 from the genome of the amphibian, Xenopus tropicalis (Xt). Heteromeric XtKir4.1/XtKir5.1 channels exhibit similar macroscopic current properties to rat Kir4.1/Kir5.1 with a faster time-dependent rate of activation. However, single channel analysis of heteromeric XtKir4.1/XtKir5.1 channels reveals that they have markedly different long-lived, multi-level subconductance states. Furthermore, we demonstrate that the XtKir5.1 subunit is responsible for these prominent subconductance levels. These results are consistent with a model in which the slow transitions between sublevel states represent the movement of individual subunits. These novel channels now provide an excellent model system to determine the structural basis of subconductance levels and contribution of heteromeric pore architecture to this process.
Supplementary Fig. 1. (A) Alignment of Kir4 sequences from rat and Xenopus tropicalis. The sequences exhibit 83% sequence identity. (B) Alignment of Kir5.1 sequences from rat and Xenopus tropicalis. The sequences exhibit 62% overall identity though this increases to 76% identity if the more divergent distal termini are excluded. The position of the βB sheet and βD/βE loops are marked. The alignment was performed using Multalin (http://bioinfo.genotoul.fr/multalin/) with highly conserved residues being coloured either red or blue according to the degree of conservation (90% and 50%, respectively).
Fig. 1. Heteromeric XtKir4.1/XtKir5.1 channels whole-cell currents. Representative whole-cell current traces from (A) rat Kir4.1/Kir5.1 channel and (B) XtKir4.1/XtKir5.1. The strong rectification and time-dependent activation are clearly visible in both cases. However, the Xt channel has a faster time-dependent activation, τ = 0.26 ± 0.09 s (n = 12) vs. τ = 2.01 ± 0.04 s (n = 9). (C) Heteromeric XtKir4.1/XtKir5.1 channels also share similar pH sensitivity and (D) Ba2+ inhibition properties to the mammalian heteromeric Kir4.1/Kir5.1 channel. The pKa of the Xt heteromeric channel, pKa = 6.90 ± 0.01 (n = 9) is very similar to that of the mammalian channel pKa = 6.8 ± 0.1 [8–10]. The currents were evoked by voltage commands from −120 to +40 mV in 10 mV increments, from a holding potential of −10 mV.
Fig. 2. The XtKir5.1 subunit is responsible for the long-lived subconductance levels. Representative traces of single-channel records of (A) ratKir4.1/ratKir5.1 (B) XtKir4.1/XtKir5.1 (C) ratKir4.1/XtKir5.1 and (D) XtKir4.1/ratKir5.1 heteromeric channels. No differences were observed in either the amplitude of the current or the ‘bursting’ single-channel behaviour with multiple subconductance states, but the subconductance states in XtKir4.1/XtKir5.1 heteromeric channels have a much longer duration. In particular, they have a long S1 sublevel opening which is almost 15 times of the average dwell time duration of the S1 sublevels observed in the rat Kir4.1/Kir5.1 channels (7.5 ms vs. 0.5 ms). RatKir4.1/XtKir5.1 heteromeric channels but not XtKir4.1/ratKir5.1 heteromeric channels also exhibit these markedly long S1 sublevels, thus confirming that the XtKir5.1 subunit is responsible for this difference.
Fig. 3. Long-lived subconducatance levels in Heteromeric Kir4.2/XtKir5.1 channels. Representative traces of single-channel records of (A) Kir4.2/Kir5.1 and (B) Kir4.2/XtKir5.1 heteromeric channels. Kir4.2/XtKir5.1 heteromeric channels also exhibited very long-lived subconductance levels as seen in the Kir4.1/XtKir5.1 channels, although in this case the S2 level is the most prominent.
Fig. 4. Heteromeric Kir4.0/XtKir5.1 channels as a model system for the study of subconductance levels. (A) Amplitude histogram of heteromeric Kir4.0/XtKir5.1 channels shows the 3 prominent subconductance levels. Arrows point to the peaks of the closed, open and S1–S3 states where individual fits are shown as dotted lines. (B) These sublevels are indicative of the proposed slower transitions of individual subunits to the fully open state in this heteromeric pore. The ability of the XtKir5.1 subunit to slow down the transitions/movements of each subunit in the heteromeric pore makes these channels a suitable model system to study the structural basis of subconductance levels.
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