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Voltage-dependent gating of SV channel TPC1 confers vacuole excitability.

ABSTRACT: In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K⁺-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca²⁺ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca²⁺, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K⁺ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K⁺-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca²⁺- and voltage-induced electrical excitability to the central organelle of plant cells.

SUBMITTER: Jaslan D

PROVIDER: S-EPMC6572840 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Voltage-dependent gating of SV channel TPC1 confers vacuole excitability.

Jaślan Dawid D Dreyer Ingo I Lu Jinping J O'Malley Ronan R Dindas Julian J Marten Irene I Hedrich Rainer R

Nature communications 20190614 1

In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K<sup>+</sup>-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca<sup>2+</sup> levels. In comparison to wild type, ...[more]

PMID: 31201323

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Project description:P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance-voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X(2)-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].

Dataset Information

Voltage-dependent gating of SV channel TPC1 confers vacuole excitability.

Publications

Voltage-dependent gating of SV channel TPC1 confers vacuole excitability.

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