title: Phosphorylation of nodulin 26 on serine 262 affects its voltage-sensitive channel activity in planar lipid bilayers.
abstract: Nodulin 26 is an symbiosome membrane protein of soybean nodules that shows ion channel activity in planar lipid bilayers. Serine 262 of nodulin 26 is phosphorylated by calmodulin-like domain protein kinase. To study the effects of phosphorylation, nodulin 26 with Ser, Ala, or Asp at position 262 were expressed in Escherichia coli. The expressed protein possessed a histidine-rich leader sequence for purification by Ni2+ chelate fast protein liquid chromatography. Upon reconstitution into planar lipid bilayers, the recombinant proteins showed a large single channel conductance (3.1 nanosiemens (nS) in cis0.2M/trans1.0 M KCl and 1.6 nS in cis 0.2M/trans0.2 M KCl) and weak anion selectivity, similar to native soybean nodulin 26. Nodulin 26 with Ser- or Ala-262 occupied the maximal open conductance state greater than 97% of the time (3.1 nS in cis0.2M/trans1.0 M KCl) regardless of applied voltage. However, nodulin 26 with Asp-262 showed increased gating and preferential occupancy of lower subconductance states (1.8 and 0.6 nS in cis0.2M/trans1.0 M KCl) at high applied voltages (e.g. 70 mV). In situ phosphorylation of Ser-262 of nodulin 26 by calmodulin-like domain protein kinase also resulted in increased voltage-dependent gating and preferential occupancy of lower subconductance states. These results suggest that phosphorylation of serine 262 of nodulin 26 modulates channel activity by conferring voltage sensitivity.
authors.affiliation: Department of Biochemistry, University of Tennessee, Knoxville 37996, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Lee
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: C D
authors.initials: CD
authors.lastname: Weaver
authors.forename: N H
authors.initials: NH
authors.lastname: Shomer
authors.forename: C F
authors.initials: CF
authors.lastname: Louis
authors.forename: D M
authors.initials: DM
authors.lastname: Roberts
mesh_terms: D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004553:Electric Conductivity; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013025:Glycine max
chemical_list: D002147:Calmodulin; D017931:DNA Primers; D018744:DNA, Plant; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C045625:nodulin; D011494:Protein Kinases