ATP-Sensitive Cation-channel in Wheat (<i>Triticum durum</i> Desf.): Identification and Characterization of a Plant Mitochondrial Channel by Patch-clamp

Abstract

Indirect evidence points to the presence of K⁺ channels in plant mitochondria. In the present study, we report the results of the first patch clamp experiments on plant mitochondria. Single-channel recordings in 150 mM potassium gluconate have allowed the biophysical characterization of a channel with a conductance of 150 pS in the inner mitochondrial membrane of mitoplasts obtained from wheat (Triticum durum Desf.). The channel displayed sharp voltage sensitivity, permeability to potassium and cation selectivity. ATP in the mM concentration range completely abolished the activity. We discuss the possible molecular identity of the channel and its possible role in the defence mechanisms against oxidative stress in plants.

Transport Pathways—Proton Motive Force Interrelationship in Durum Wheat Mitochondria

“…Its existence was assessed firstly by using swelling technique and by following channel-dependent ΔΨ changes [44], and it has been successively confirmed by means of the first patch clamp study on plant mitochondria [45]. In these mitochondria, K + uptake is mediated by an ATP-inhibited channel named plant mitoK ATP (PmitoK ATP ) by analogy to the possible animal counterpart, the mitoK ATP [46].…”

A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

“…In fact, in experiments measuring ∆Ψ changes due to K + uptake, ATP inhibited the channel in a non-competitive manner with a K i of about 290 µM (Pastore et al 1999); moreover, a K 0.5 equal to 0.5 mM ATP was measured in patch clamp experiments (De Marchi et al 2010). Since K m for K + is 2.2 mM (Pastore et al 1999), and considering that mitochondria live in an ionic cytoplasm containing about 100 mM K + and 0.6-0.8 mM ATP (see above), the degree of channel activity, according to the Michaelis-Menten equation, should be about 26-44% of V max in control condition, whereas the measured drop of ATP under our stress conditions may increase channel activity up to about 90%.…”

“…Moreover, a mitochondrial K + uniport has been reported in many other plant species, including bread wheat, barley, spelt, rye, spinach (Pastore et al 1999), potato (Pastore et al 1999;Fratianni et al 2001), and triticale, topinambur and lentil . Recently, the first patch clamp experiments on DWM have identified an ATP-inhibited K + channel also at the level of single channel activity (De Marchi et al 2010). At the same time, a channel with similar single channel and pharmacological properties was observed in bilayer experiments using plant mitochondria inner membrane vesicles (Matkovic et al 2011).…”

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

“…It will be possible to clarify the exact link between the lack of AtGLR3.5, the mitochondrial changes, and the anticipated senescence only once the nature of the ions flowing through this protein and the regulation of channel activity are understood. Unfortunately, as mentioned, our attempts to perform patch-clamp experiments on isolated Arabidopsis mitochondria using a protocol that was previously successfully applied for wheat (Triticum aestivum) germ mitochondria (De Marchi et al, 2010) failed. In addition, attempts to record channel activity using the cell-free expressed protein incorporated into an artificial membrane performed as described previously (De Stefani et al, 2011) suggest that an additional, unknown component is necessary to reconstitute the functional AtGLR3.5 channel.…”

“…After centrifugation (10 s at 3,000g at 4°C), the supernatant was centrifuged for 15 min at 10,000g to collect the mitochondria (De Marchi et al, 2010).…”