Abstract
Two members of the family of high conductance K(+)channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca(2+)and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na(+) Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn(2+)being the most effective inhibitor with an IC50of ∼8 μmin contrast to Mg(2+), the least effective, with an IC50of ∼ 1.5 mm Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel fromDrosophila(dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na(+)and inhibited by divalent cations. Inhibition of SLO2 channels in mammals andDrosophilaby divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions.
Highlights
Potassium (Kϩ)2 channels of the SLO family are the largest and most complex Kϩ channels [1, 2]
To investigate possible structural features in SLO2.2 channels involved with divalent cation inhibition we examined a comparative alignment of SLO2.2 with cyclic nucleotide-activated channels known to be inhibited by divalent ions [19]
It had been reported that calcium ions inhibit native Naϩ-dependent Kϩ channels, likely to be SLO2.2, in thick ascending limb of mouse kidney [20] but the effect of calcium has not been confirmed in heterologously expressed SLO2.2 channels
Summary
Potassium (Kϩ)2 channels of the SLO family are the largest and most complex Kϩ channels [1, 2]. Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. These results contrast with the effects of Ba2ϩ on SLO1 channels which has been reported to both activate and inhibit SLO1 channels, with the latter showing an obvious voltage-dependent pore-blocking effect [18].
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