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A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

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Summary

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts ofVicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. “Laserassisted” patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

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Abbreviations

CAP:

cell-attached patch

E0 :

membrane potential

E k :

Nernst potential for K+

Ec :

true reversal potential of the channel in CAP

I k,out :

outwardly rectifying K+ channel

LJP:

liquid junction potential

Vm :

actual applied potential

Vp :

pipette potential

V op :

pipette potential at zero observed current

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Author notes

  1. Hendrik Miedema

    Present address: Instituto de Biotecnologia, Universidad Nacional Autónoma de Mexico, Cuernavaca, Morelos, Mexico

Authors and Affiliations

  1. Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, 16802, University Park, PA, USA

    Hendrik Miedema, Gordon H. Henriksen & Sarah M. Assmann

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  1. Hendrik Miedema
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  2. Gordon H. Henriksen
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  3. Sarah M. Assmann
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Miedema, H., Henriksen, G.H. & Assmann, S.M. A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane. Protoplasma 209, 58–67 (1999). https://doi.org/10.1007/BF01415701

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  • DOI: https://doi.org/10.1007/BF01415701

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