Hosy E et al. (2005), A procedure for localisation and electrophysiol...

XB-ART-930

Plant Methods 2005 Dec 19;1:14. doi: 10.1186/1746-4811-1-14.

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A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.

Hosy E , Duby G , Véry AA , Costa A , Sentenac H , Thibaud JB .

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In silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels. With the aim of developing a plant expression system readily amenable to electrophysiological analyses, we optimised experimental conditions for preparation and transformation of tobacco mesophyll protoplasts and engineered expression plasmids, that were designed to allow subcellular localisation and functional characterisation of ion channels eventually in presence of their putative (possibly over-expressed) regulatory partners. Two inward K+ channels from the Shaker family were functionally expressed in this system: not only the compliant KAT1 but also the recalcitrant AKT1 channel, which remains electrically silent when expressed in Xenopus oocytes or in mammalian cells. The level of endogenous currents in control protoplasts seems compatible with the use of the described experimental procedures for the characterisation of plant ion channels, by studying for instance their subcellular localisation, functional properties, structure-function relationships, interacting partners and regulation, very likely in a more realistic context than the classically used animal systems.

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Species referenced: Xenopus
Genes referenced: acta2 akt1 kyat1

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	Figure 1. Expression vectors engineered for transformation of tobacco mesophyll protoplasts. (A) pLoc. This vector carries a single over-expression cassette. The green fluorescent protein cDNA ("GFP") is framed by a strong promoter ("EN50PMA4") and a terminator ("T"). Three restriction sites (Bgl II, BamH I, Kpn I) are available for in-phase cloning of another cDNA. "ATG" indicates the first GFP codon. This vector allows the expression of fusion polypeptides with a GFP-tag in C-terminal for localisation purpose. (B) pFunct+Tag. This vector carries two expression cassettes both featuring the EN50PMA4 promoter and the T terminator. The first cassette encodes the GFP alone and the second one displays two restriction sites (Xho I, Sma I) for cDNA cloning. pFunct+Tag is used for functional expression of the recombinant cDNA while fluotagging transformed cells. (C) pFunct. This vector has the same construction as pFunct+Tag but the GFP cassette is absent. It is used, in combination of pLoc or pFunct+Tag, when two cDNAs are to be co-expressed (see text). EN50PMA4 is a tobacco enhanced promoter (see "Methods"); and T: is the nopaline synthase terminator of Agrobacterium tumefaciens. These vectors were obtained in a pTZ-19U plasmid (Stratagene, LaJolla, CA, USA) background.
	Figure 2. Native K+ and Cl- currents in tobacco mesophyll protoplasts transformed by the empty pLoc vector. (A, B) Typical recordings illustrating the two patterns of whole-cell inward K+ currents elicited by membrane hyperpolarisation. 35 % of the patch-clamped protoplasts displayed the "no-current" pattern shown in (A). 65 % of the patch-clamped protoplasts displayed the voltage-dependent instantaneous weak current pattern shown in (B). (C) Typical recordings of whole-cell outward K+ currents elicited by membrane depolarisation on the same protoplasts as in (A) and (B). The voltage steps ranged from -60 mV to -200 mV (A, B) and from -40 mV to +100 mV (C) in +20 mV increments, from a holding potential of -40 mV. The symbol above the records in a-c indicates the time of "steady-state" current sampling. (D) Average (mean ± SE, n = 10) of native steady-state K+ currents in tobacco mesophyll protoplasts plotted against membrane potential. (E) Typical recordings of native whole-cell Cl- currents recorded in protoplasts exposed to CsCl in pipette and extracellular solutions (see "Methods"). The voltage steps ranged from -202 mV to +58 mV in +20 mV increments and the holding potential was -22 mV. Dashed line marks zero current level. The symbol above the records indicates the time of "steady-state" current sampling. (F) Average (mean ± SE, n = 10) of native steady-state Cl-currents in tobacco mesophyll protoplasts plotted against membrane potential. Voltage dependence, at steady state, of the native chloride currents in tobacco mesophyll protoplasts (means ± SE; n = 10). ECl, ECs and ECa represent equilibrium potentials for Cl-, Cs+ and Ca2+ respectively (see detailed content of bath and pipette solutions in "Methods").
	Figure 3. Functional expression and subcellular localisation of AKT1 and KAT1 channels in tobacco mesophyll protoplasts. (A) and (C) Typical recordings of the whole-cell inward and outward K+ currents in patch-clamped tobacco mesophyll protoplasts respectively transformed with AKT1-carrying and KAT1-carrying pFunct+Tag vectors. The voltage steps ranged from -200 mV to +100 mV in 20 mV increments. The holding potential was 0 mV and -40 mV respectively for AKT1 and KAT1 expressing protoplasts. The symbol above the records in a and c indicates the time of "steady-state" current sampling. (B) and (D) Current-voltage relationships at steady state in control tobacco mesophyll protoplasts (closed circles in both B and D) and in AKT1-expressing (open squares in B) and KAT1-expressing (open circles in d) ones (means ± SE; n = 16 for AKT1, n = 13 for KAT1). (E, F) Confocal microscopy sections of protoplasts transformed with AKT1-carrying (E) and KAT1-carrying (F) pLoc vectors. The left panels display protoplast sections analysed for the GFP fluorescence, the middle panels the same sections analysed for chloroplast auto-fluorescence and FM4-64 fluorescence and the right panels the overlay of the two former panels with the transmission light image from the same protoplast section. FM4-64 was 50 μM in both (E) and (F) and was incubated for 10 min on ice in (E) and for 40 min at room temperature in (F). Some places where GFP and FM4-64 fluorescence co-localises are marked by white arrows in (F). Scale bar = 20 μm.
	Figure 4. Windowing the time allowed for patch-clamp recordings. (A) Confocal microscopy sections of tobacco mesophyll protoplasts transformed with empty "localisation" plasmids (control protoplasts). Left panel: protoplast analysed for GFP detection. Middle and right panels: images of protoplasts bathing in calcofluor dye solution respectively without and with wall. Bar = 20 μm. (B) Time-course of GFP expression in transformed tobacco mesophyll protoplasts. The number of cells displaying GFP expression at a given time after transformation is expressed as a percentage of the number of cells which finally (55 hours after transformation) expressed GFP. Closed symbols: 3 independent transformations with empty pLoc ("GFP"). Open symbols: 2 independent transformations with pFunct+Tag-KAT1 ("channel+GFP"). About 500 transformed cells were considered for each experiment. Line represents exponential fit of the data. (C) Time-course of cell wall regeneration. The cell wall was marked with calcofluor dye. A cell was considered to have a wall if part of its surface showed blue staining. Each point represents about 200 protoplasts. Triangles and circles represent protoplasts transformed respectively with empty pLoc and pFunct+Tag-KAT1. Dark symbols represent all the protoplasts and open symbols those displaying green fluorescence. Line represents sigmoidal fit of the data. (D) Operational time window for patch-clamp recordings. Superimposition of the GFP apparition and cell wall synthesis fitted curves allows determination of a time frame for patch-clamp experiments (see text). (E) Time-course of the amplitude of steady-state currents recorded at -200 mV in tobacco mesophyll protoplasts transformed with pFunct+Tag-KAT1 (dark symbols) or pFunct+Tag-AKT1 (open symbols).

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Ache, GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K(+)-selective, K(+)-sensing ion channel. 2000, Pubmed, Xenbase