Commandeering Channel Voltage Sensors for Secretion, Cell Turgor, and Volume Control

Abstract

Control of cell volume and osmolarity is central to cellular homeostasis in all eukaryotes. It lies at the heart of the century-old problem of how plants regulate turgor, mineral and water transport. Plants use strongly electrogenic H⁺-ATPases, and the substantial membrane voltages they foster, to drive solute accumulation and generate turgor pressure for cell expansion. Vesicle traffic adds membrane surface and contributes to wall remodelling as the cell grows. Although a balance between vesicle traffic and ion transport is essential for cell turgor and volume control, the mechanisms coordinating these processes have remained obscure. Recent discoveries have now uncovered interactions between conserved subsets of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that drive the final steps in secretory vesicle traffic and ion channels that mediate in inorganic solute uptake. These findings establish the core of molecular links, previously unanticipated, that coordinate cellular homeostasis and cell expansion.

Keywords: K(+) channels; SNARE protein; Sec1-Munc18 protein; plant cell turgor; secretion; voltage-dependent; volume control.

Figure 1

The SNARE Cycle. SNAREs assemble a tetrameric coiled-coil around a central core of three glutamine (Qa, Qb, and Qc) and one arginine (R) residues, in 1:1:1:1 stoichiometry, to draw vesicle and target membranes together for fusion. Each motif is defined by the amino acid at the core of the interacting hydrophobic layers between the respective peptide coils. In some cases, the Qb- and Qc-SNARE motifs reside on a single polypeptide, as is the case for the arabidopsis SNAP33 protein, but, more commonly, each motif in a SNARE complex is contributed by a separate protein , , , , . (A) Binding of the membrane- and vesicle-localised SNAREs in a cognate SNARE core complex involves priming of the Qa-SNARE at the target membrane. The Qa-SNARE transits from its ‘closed’ to ‘open’ conformation through the unfurling of the Habc domain, which exposes the Qa-SNARE domain for binding with the Qb- and Qc-SNAREs. The Qa-, Qb-, and Qc-SNAREs when bound together form the ‘acceptor’ complex, which is available for binding to the R-SNARE. (B) Formation of the trans-SNARE complex following interactions between the R-SNARE on the vesicle and the ‘acceptor’ complex at the target membrane draws the secretory vesicle to the target membrane for fusion. (C) Formation of a stabilised Qa-, Qb-, Qc-, and R-SNARE core complex overcomes the hydration energy barrier of the membrane surfaces and leads to fusion of the secretory vesicle with the target membrane and release of the vesicle cargo. (D) Following vesicle fusion, cis-SNARE complex disassembly allows for recycling of the SNARE proteins and sustains further secretory traffic. Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TM, transmembrane.

Figure 2

Complementarity of SYP121^ΔC and the Kv Channel Voltage-Sensor Domain Is A Predictable Consequence of Their Dual Functionalities. K⁺ channels are shown with orange pore domains and purple voltage-sensor domains (VSDs). SYP121 is shown in red with membrane anchor, longer Qa-SNARE domain, and shorter Habc domain; the dominant-negative SYP121^ΔC is shown lacking the membrane anchor; VAMP721 is shown in blue with a membrane anchor, longer R-SNARE domain and a shorter longin domain. The FxRF motif of SYP121 with the VSD is shown as a circle at the SYP121 N terminus. Other SNARE proteins have been omitted for clarity. Comparison of the relative rates for vesicle traffic and solute influx are represented by bar graphs (right). (A) In the absence of either dominant-negative fragment, SNARE binding with the K⁺ channels facilitates the activation and balance of both processes. (B) Overexpressing the channel VSD facilitates SNARE-mediated traffic, but competes with the native channels for SNARE binding, thereby suppressing K⁺ influx and its accumulation. (C) Overexpressing the SYP121^ΔC fragment facilitates K⁺ flux by binding and activating the K⁺ channels, but the SYP121^ΔC fragment competes with the native SNARE at the plasma membrane for assembly in the SNARE complex needed to drive vesicle fusion. Thus, in each case, the dominant-negative fragment uncouples vesicle traffic and K⁺ uptake with complementary effects on the two processes. Abbreviation: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.

Figure 3

Voltage Alters the Exposure of the Kv Channel Voltage Sensor to the Cytosol. Side views of the voltage sensor domain (VSD) of the KAT1K⁺ channel are shown in the ‘up’ (A) and ‘down’ (B) conformations. For clarity, the membrane bilayer is not shown and only water molecules (light blue) on either side (inside and outside) of the membrane are visible. Transmembrane α-helices are colour coded in green (S1), black (S2), red (S3), and yellow (S4). Residues RYxxWE that form the binding site for SYP121 are included in blue as stick representations. The molecular dynamics of each conformation were modelled using as a template the entire α-subunit of Kv1.2 obtained by the Rosetta method , . Each model was analysed after embedding within a lipid bilayer in a periodic boundary condition box with water molecules, K⁺ and Cl^– ions, and was optimised using energy minimisation and equilibration at 298 K for 5 ns with a harmonic restraint of 0.5 kcal/mol Å² applied to the backbone atom using NAMD . As the membrane is hyperpolarised, movement of the S4 domain within the transmembrane electric field drives the transition to the ‘down’ conformation and is coupled to rotation of the S5 and S6 α-helices to open the channel pore (not shown). Here, the ‘down’ conformation corresponds to the open channel. On the basis of simulations , it appears also to promote the aqueous exposure of the RYxxWE motif at the cytosolic surface of the membrane. Images created with VMD .

Figure 4

A Model for Kv Channel-Binding Exchange between VAMP721 and SYP121. K⁺ channels are shown with orange pore domains and purple voltage-sensor domains (VSDs). SYP121 is shown in red with membrane anchor, longer Qa-SNARE domain, and shorter Habc domain with the FxRF motif shown as a circle and Phe⁹ highlighted; VAMP721 is shown in blue with membrane anchor, longer R-SNARE domain, and shorter longin domain with Tyr⁵⁷ indicated. SEC11 is depicted in green with the major cleft formed by the black line and residue Leu¹²⁸, important for SEC11 interactions with SYP121 , , is indicated near the N-terminal surface. Other SNARE proteins have been omitted for clarity. Voltage is denoted by charges (+ and –), with membrane hyperpolarisation indicated with increased negative charge inside the cell. (A) SYP121 in its ‘closed’ conformation is stabilised by SEC11 bound to the SYP121 N terminus with the interaction with Leu¹²⁸ in the SEC11 minor cleft essential for unfurling of the SYP121 Habc domain. (B,C) VAMP721 on the secretory vesicle binds the closed K⁺ channel to help recruit the vesicle to the plasma membrane. (D) VAMP721 and the open conformation of SYP121 exchange channel binding, facilitated by membrane hyperpolarisation and channel opening with VSD in the ‘down’ state, assembling the trans-SNARE complex, which is stabilised by SEC11. (E) SEC11 facilitates release from channel and disassembly of the cis-SNARE complex. (F) SNARE complex disassembles to recycle the cognate SNAREs for subsequent fusion events. Abbreviation: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.

Figure I

Total Membrane IV Curves and Their Decomposition.