A type of voltage-dependent Ca²⁺ channel on Vicia faba guard cell plasma membrane outwardly permeates K⁺

Abstract

The fine regulation of stomatal aperture is important for both plant photosynthesis and transpiration, while stomatal closing is an essential plant response to biotic and abiotic stresses such as drought, salinity, wounding, and pathogens. Quick stomatal closing is primarily due to rapid solute loss. Cytosolic free calcium ([Ca²⁺]_cyt) is a ubiquitous second messenger, and its elevation or oscillation plays important roles in stomatal movements, which can be triggered by the opening of Ca²⁺-permeable channels on the plasma membrane. For Ca²⁺-permeable channel recordings, Ba²⁺ is preferred as a charge-carrying ion because it has higher permeability to Ca²⁺ channels and blocks K⁺ channel activities to facilitate current recordings; however, it prevents visualization of Ca²⁺ channels’ K⁺ permeability. Here, we employed Ca²⁺ instead of Ba²⁺ in recording Ca²⁺-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Inward Ca²⁺ currents could be recorded at the single-channel level, and these currents could be inhibited by micromolar Gd³⁺, but their reversal potential is far away from the theoretical equilibrium potential for Ca²⁺. Further experiments showed that the discrepancy of the reversal potential of the recorded Ca²⁺ currents is influenced by cytosolic K⁺. This suggests that voltage-dependent Ca²⁺ channels also mediate K⁺ efflux at depolarization voltages. In addition, a new kind of high-conductance channels with fivefold to normal Ca²⁺ channel and 18-fold to normal outward K⁺ conductance was found. Our data presented here suggest that plants have their own saving strategies in their rapid response to stress stimuli, and multiple kinds of hyperpolarization-activated Ca²⁺-permeable channels coexist on plasma membranes.