| Abstract: | Microsomal membranes from rye (Secale cereale L.) roots wereseparated by isopycnic sucrose density gradient centrifugation.The ion channels present in gradient fractions were assayedby reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolaminebilayers (PLB) and the distributions of ion channel activitieswere compared with membrane markerenzyme activities. A numberof ion channel activities were observed and could be distinguishedon the combined bases of their conductance, selectivity, kineticsand pharmacology. A voltage-dependent maxi (498 pS) cation-channel,a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K+channels, and a 148-pS Cl– channel (all unitary conductancesdetermined in asymmetrical cis trans 325:100mM KCl) colocalizedwith the plasma membrane marker-enzyme, vanadatesensitive ATPase.A weakly K +-selective (108 pS) channel, a 1249-pS cation-channeland a 98-pS K + channel colocalized with the tonoplast markerenzyme,nitrate-sensitive ATPase. A 706-pS K+ channel colocalized withthe expected distribution of intact plastids and a 38-pS Cl–channel colocalized with either plastid or ER membranes. Themembrane location of several other channels including a hypervoltage-sensitivemaxi (497 pS) cation-channel, a 270-pS K+ channel, an 8-pS K+channel and a 4-pS K+ channel was equivocal, but they were tentativelyassigned to the Golgi. Thus, the plasma membrane and tonoplastorigin of ion channels previously characterized following theincorporation of plasma membrane prepared by aqueous-polymertwo-phase partitioning or tonoplast derived from isolated vacuolesinto PLB was confirmed and the ion channel complement of previouslyunassayed membranes was defined. This demonstrates the usefulnessof PLB in identifying and characterizing ion channels from plantcell membranes, in particular, those of membranes which areinaccessible to patch-clamp electrodes. Key words: Chloride (Cl–) channel, potassium (K+) channel, planar lipid bilayer, root, rye, Secale cerealeL. |
