Calcium‐regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts

Abstract

Summary• Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration.• With Cl ) -based intra-and extracellular solutions, three outward-rectifying anion conductances, I Cl1 , I Cl2 and I Cl3 , were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca 2+ ] in ), increasing intensity from 6.04 nM up to 0.5 mM (I Cl1 ), or reaching a maximum value with 8.50 lM (I Cl2 and I Cl3 ).• After rundown, the anionic currents measured using NO 3 ) -based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl ) and NO 3 ) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels.• This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca 2+ -regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes.

Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20

“…Similar to patch-clamp analysis of anion currents in lily pollen tube protoplasts (Tavares et al, 2011a), our analysis revealed depolarization-activated, slowly deactivating currents in wild-type Columbia-0 (Col-0), whereas typical S-type anion currents were absent or strongly reduced in slah3-1, slah3-2, and cpk2-1 cpk20-2 mutant cells ( Figure 7B). A plot of the steady state currents from membrane voltages clamped from 2200 mV to +80 mV (D = 20 mV) revealed significantly reduced anion currents in slah3-1-, slah3-2, and cpk2-1 cpk20-2 pollen tubes in both chloride-based (see Supplemental Figure 12 online) and nitratebased solution, pointing to a function for CPK2 and CPK20 in S-type anion channel regulation ( Figure 7C).…”

“…This cytosolic anion gradient matched the plasma membrane anion fluxes, as described by Zonia et al (2002), and currents, as described by Tavares et al (2011a). The cytosolic gradient was characterized by high chloride concentration in a cytosolic domain ranging from ;20 to ;40 µm behind the tip.…”

“…Subsequent studies by patch clamp of pollen protoplasts suggest that these fluxes are derived from [Ca 2+ ] cyt activated anion channel activity in lily (Tavares et al, 2011a). Thus far, however, the molecular nature and mechanisms of the pollen tube anion transporters and regulators are largely unknown.…”

“…Inhibition of anion fluxes in pollen tubes or grains via inhibitors results in growth arrest or retains pollen germination, respectively; furthermore, pharmacological and osmotic manipulations suggest that anion effluxes have a crucial role in water homeostasis (Zonia et al, 2002;Breygina et al, 2010). Patch-clamp studies revealed three different anion channel conductances in pollen grains and tube protoplasts (Tavares et al, 2011a).…”

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels inArabidopsisPollen Tubes

“…In guard cells, Ca 2+ signals can be transduced from some CDPKs or other Ca 2+ sensor proteins to various types of ion channels, such as K + in channels, nonselective Ca 2+ -permeable channels, and S-type anion channels, to regulate stomatal movements (Schroeder and Hagiwara, 1989;Li et al, 1998;Berkowitz et al, 2000;Mori et al, 2006;Geiger et al, 2010;Zou et al, 2010). Recently, Cl 2 channels and outward K + channels were reported to be regulated by intracellular Ca 2+ in pollen tubes (Tavares et al, 2011;Wu et al, 2011).…”