Biochemical, molecular and functional characterization of plant ion channels

A rice cDNA encoding a 36.3 kD polypeptide (KOB1), which is homologous to the cloned animal and plant {dollar}{bsol}rm K{bsol}sp+{dollar} channel {dollar}{bsol}beta{dollar} subunits, has been cloned. The KOB1 translation product was demonstrated to form a tight physical association with a plant {dollar}{bsol}rm K{bsol}sp+{dollar} channel {dollar}{bsol}alpha{dollar} subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a {dollar}{bsol}rm K{bsol}sp+{dollar} channel {dollar}{bsol}beta{dollar} subunit. Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when {dollar}{bsol}rm K{bsol}sp+{dollar} was lost from older leaves of plants grown on {dollar}{bsol}rm K{bsol}sp+{dollar}-deficient fertilizer, the loss of cellular {dollar}{bsol}rm K{bsol}sp+{dollar} was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration that changes in cellular {dollar}{bsol}rm K{bsol}sp+{dollar} status may specifically alter expression of a gene encoding a {dollar}{bsol}rm K{bsol}sp+{dollar} channel subunit.; Transport studies identified a {dollar}{bsol}rm K{bsol}sp+{dollar} channel protein in spinach thylakoid membrane. This protein was solubilized from native membranes and reconstituted into artificial proteoliposomes with maintenance of functional integrity. A 33 kD thylakoid polypeptide was identified as a putative component of this thylakoid protein. {dollar}{bsol}rm K{bsol}sp+{dollar} channel activity co-migrated with the immunoreactive 33 kD polypeptide. The antibody was used to immunoprecipitate the 33 kD polypeptide. Studies of physiological function of this thylakoid membrane protein indicated that {dollar}{bsol}rm K{bsol}sp+{dollar} efflux from the thylakoid lumen through this channel protein is required for the optimization of photosynthetic capacity.; A cDNA encoding a VDAC-type ion channel protein from spinach (SVDAC1) was cloned. Research results show that SVDAC1 gene encodes a polypeptide 276 amino acids long with a deduced molecular mass of 29.7 kD. This primary protein sequence shares little homology with yeast and animal VDAC protein sequences. However, the amino acid identity between SVDAC1 and seven other cloned plant VDACs is quite high. Protein import assay indicates that the SVDAC1 protein associates with the (outer) membrane of mitochondria.