Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor

The Kv-like K+ channels at the plasma membrane, including the inward-rectifying KAT1 K+ channel of Arabidopsis, are important targets for manipulating K+ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K+ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-mutagenesis to explore residues that are thought to form two electrostatic counter-charge centers either side of a conserved Phe residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the counter-charge centers favored the open channel. Modelling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in context of the effects on hydration of amino-acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel

Modulation of plant TPC channels by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are powerful modulators of several animal ion channels. It is shown here that PUFAs strongly affect the activity of the Slow Vacuolar (SV) channel encoded by the plant TPC1 gene. The patch-clamp technique was applied to isolated vacuoles from carrot taproots and Arabidopsis thaliana mesophyll cells and arachidonic acid (AA) was chosen as a model molecule for PUFAs. Our study was extended to different PUFAs including the endogenous alpha-linolenic acid (ALA). The addition of micromolar concentrations of AA reversibly inhibited the SV channel decreasing the maximum open probability and shifting the half activation voltage to positive values. Comparing the effects of different PUFAs, it was found that the length of the lipophilic acyl chain, the number of double bonds and the polar head were critical for channel modulation.The experimental data can be reproduced by a simple three-state model, in which PUFAs do not interact directly with the voltage sensors but affect the voltage-independent transition that leads the channel from the open state to the closed configuration. The results indicate that lipids play an important role in co-ordinating ion channel activities similar to what is known from animal cell

Modulation of plant TPC channels by polyunsaturated fatty acids

Poyunsaturated fatty acids (PUFAs) are powerful modulators of several animal ion channels. It is shown here that PUFAs strongly affect the activity of the Slow Valcuolar (SV) channel encoded by the plant TPC1 gene. The patchclamp technique was applied to isolated vaculoes from carrot taproots and Arabidopsis thaliana mesophyll cells and arachidonic acid (AA) was chosen as a model molecule for PUFAs. Our study was extended to different PUFAs inhibited the SV channel decreasing the maximum opne probability and shifting the half activation voltage to positive values. Comparing the effects of different PUFAs, it was found that the length of the lipophilic acyl chain, the number of double bonds and the polar head were critical for channel modulation. The experimental data can be reproduced by a simple three-state model, in which PUFAs do not interact directly with the voltage sensors but affect the vltage independent transition that leads the channel from the open state to the closed configuration. The results indicate that lipids play an important role in co-ordinating ion channel activities similar to what is known from animal cells. \ua9 2012 The Author