A mechanosensitive Ca2+ channel activity is dependent on the developmental regulator DEK1

Abstract

Responses of cells to mechanical stress are thought to be critical in coordinating growth and development. Consistent with this idea, mechanically activated channels play important roles in animal development. For example, the PIEZO1 channel controls cell division and epithelial-layer integrity and is necessary for vascular development in mammals. In plants, the actual contribution of mechanoperception to development remains questionable because very few putative mechanosensors have been identified and the phenotypes of the corresponding mutants are rather mild. Here, we show that the Arabidopsis Defective Kernel 1 (DEK1) protein, which is essential for development beyond early embryogenesis, is associated with a mechanically activated Ca²⁺ current in planta, suggesting that perception of mechanical stress plays a critical role in plant development.

Fig. 1

A mechanically activated current permeable to Ca²⁺ is present at the plasma membrane. a Representative membrane patches from Col-0 (left) and an msl quintuple mutant msl4;msl5;msl6;msl9;msl10 (mslΔ5, right), exposed to increased positive pressure steps in an outside-out patch configuration show a rapidly activated, rapidly inactivated current in ionic conditions favorable for Ca²⁺ current recording. We have named the channel responsible for this activity the RMA channel (see text). Time constants are means ± SE (n = 6); b Under the same conditions, single channel I/V curves show similar RMA channel conductance in Col-0 (solid square) and in the mslΔ5 mutant (open square). Values are means ± SE (n = 6); c Representative single channel recordings showing that the RMA current is reversibly eliminated by exchanging Ca²⁺ ions with non-permeant TEA⁺ ions. d Open probability (for n channels, (nP(o)) is severely decreased in TEA⁺ bath solution. A paired t-test was used to compare means (**P < 0.01). Values are means ± SE. For all experiments, the membrane potential was held at −196 mV. Ionic conditions are described in the Methods section

Fig. 2

The mechanically activated RMA current is preferentially permeable to Ca²⁺ and affected by both the mechanosensitive channel inhibitor Gd³⁺ and the calcium channel blocker La³⁺. a Representative single channel recordings in response to positive pressure, in an outside-out patch configuration with Ca²⁺ (top) or Ba²⁺ (bottom) as the permeant bivalent cation in the bath. The dotted lines indicate the open (O) and closed (C) channel state; b Single channel I/V curves show similar conductance with permeant cations Ca²⁺ (solid square) and Ba²⁺ (open square). Values are means ± SE (n = 6); c The mechanically activated RMA current is inhibited by Gd³⁺ and restored after wash-out (left, n = 6) and the corresponding open probability (nP(o), right). Values are means ± SE; d Representative recording showing the effect of La³⁺ (left, n = 6) on the RMA current; e La³⁺ ions affects the open state of the RMA channel and reducing the mean open time. Bars represent the two time constant τ₁ and τ₂ of the open state with (white bars) or without (black bars) La³⁺. A paired t-test was used to compare means (P < 0.01). E_rev(Ca²⁺) = 47.5 ± 5.4 mV; E_rev(Ba²⁺) = 36.5 ± 6.1 mV. Values are means ± SE (open events, n ≥ 100). For all experiments, the membrane potential was held at −196 mV. Ionic conditions are described in the Methods section

Fig. 3

RMA activation depends on the trans-membrane region of the essential DEK1 protein. a Mechanosensitive current kinetics in response to pulse pressure (as illustrated) in dek1-2 CALPAIN-OE, dek1-3 CALPAIN-OE, Col-0 CALPAIN-OE, dek1-2 DEK-GFP and dek1-3 DEK-GFP. Time constants are means ± SE (n = 6); b Inactivation times are fitted with an exponential function, and time constants (τ_inact) are represented in a box plot distribution for different lines. Number in brackets represent the number of time constants. *significantly different from Col-0 line, Anova on ranks (P < 0.05). For all experiments the membrane potential was held at −196 mV. Ionic conditions are described in the Methods section