Patch clamp study of mechanism of membrane depolarization induced by a pulse of blue light in pulvinar motor cells of Phaseolus vulgaris

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1. Phaseolus vulgaris L. was cultivated under light regime of 12h light/12h dark at relative humidity of 70% and 23℃. Materials of 2-3 weeks were usually used for preparing protoplasts. 2. Measurements of both apoplastic concentration of KィイD1+ィエD1 and apoplastic potential with double-barreled KィイD1+ィエD1 microelectrodes revealed close relationship between depolarization of membrane potential and efflux of KィイD1+ィエD1 from motor cells of pulvinus. 3. Protoplasts of pulvinar motor cells were prepared mainly according to methods taken by Wang & Iino (1998) and Vogelzhang & Prins (1992). 4. Seal tests of patch electrodes against tonoplast prepared from protoplasts with hypotonic treatment were tried and successful (more than 1GΩ). That showed the system for performing patch-clamping works without problems. 5. Seal tests of patch electrodes against plasmalemma of protoplasts were not successful (less than 0.1GΩ) though following points were tried to get giga seal. Days for cultivating materials, osmotic and potential differences between patch pipettes and external media, quality of glass of pipettes, resistances of electrodes, preparing protoplasts without debris of cell walls on the surface of plasmalemma. The last point gave maximum seal resistance of patch electrode (0.1GΩ). As a result, patch clamp study of involvement of plasmalemma HィイD1+ィエD1-pump on blue-light-induced membrane depolarization could not be performed. 6. Biochemical assay of HィイD1+ィエD1-pump activity using suspension of protoplasts is now being undertaken indirectly to confirm whether or not HィイD1+ィエD1-pump of plasmalemma is inactivated during irradiation of blue light.