Phosphorylation of nodulin 26 by calmodulin-like domain protein kinase confers voltage-sensitivity in planar lipid bilayers

Author

Jung Weon Lee

Date of Award

8-1995

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

Jim Hall, John Koontz

Abstract

Nodulin 26 is an integral symbiosome membrane protein of soybean nodules. Purified nodulin 26 shows a voltage-sensitive, high conductance ion channel activity upon reconstitution into planar lipid bilayers. Ser 262 of nodulin 26 is phosphorylated by calmodulin-like domain protein kinase (CDPK). To study the effects of phosphorylation on channel activity, recombinant nodulin 26 with ser, ala, or asp at residue 262 were expressed from nodulin 26 cDNAs in E. coli. The expressed protein possessed an amino terminal histidine-rich sequence that allowed purification by FPLC Ni²⁺-chelate Upon reconstitution into planar lipid bilayers, the recombinant proteins showed channel activity with a large unitary conductance (3.1 nanosiemens [nS] in cis_{0.2 M}/trans_{1.0 M KCl} and 1.6 in cis_{0.2 M}/trans_{0.2 M KCl}) and weak anion selectivity, similar to native soybean nodulin 26. chromatography. Recombinant nodulin 26 with ser or ala 262 remained completely open (3.1 nS in cis_{0.2 M}/trans_{1.0 M KCl}) at high applied voltages (e.g., 70 mV). However, recombinant nodulin 26 with open asp 262 showed increased voltage dependent gating and a preference for lower subconductance states (3.1, 1.8, and 0.6 nS in cis_{0.2 M}/trans_{1.0 M KCl}) at 70 mV. In situ phosphorylation of the nodulin 26 channel at ser 262 by CDPK resulted in similar increases in voltage-dependent gating and the population of lower subconductance states. Voltage- independent behavior was restored by dephosphorylation with alkaline phosphatase. These results suggest that phosphoryla-tion at ser 262 of nodulin 26 by CDPK modulates the sensitivity of the channel to high applied voltages, and may constitute an inactivation gate.

Recommended Citation

Lee, Jung Weon, "Phosphorylation of nodulin 26 by calmodulin-like domain protein kinase confers voltage-sensitivity in planar lipid bilayers. " Master's Thesis, University of Tennessee, 1995.
https://trace.tennessee.edu/utk_gradthes/11170