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Interactions between the C-terminus of Kv1.5 and Kvβ regulate pyridine nucleotide-dependent changes in channel gating

  • Ion Channels, Receptors and Transporters
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Abstract

Voltage-gated potassium (Kv) channels are tetrameric assemblies of transmembrane Kv proteins with cytosolic N- and C-termini. The N-terminal domain of Kv1 proteins binds to β-subunits, but the role of the C-terminus is less clear. Therefore, we studied the role of the C-terminus in regulating Kv1.5 channel and its interactions with Kvβ-subunits. When expressed in COS-7 cells, deletion of the C-terminal domain of Kv1.5 did not affect channel gating or kinetics. Coexpression of Kv1.5 with Kvβ3 increased current inactivation, whereas Kvβ2 caused a hyperpolarizing shift in the voltage dependence of current activation. Inclusion of NADPH in the patch pipette solution accelerated the inactivation of Kv1.5-Kvβ3 currents. In contrast, NADP+ decreased the rate and the extent of Kvβ3-induced inactivation and reversed the hyperpolarizing shift in the voltage dependence of activation induced by Kvβ2. Currents generated by Kv1.5ΔC+Kvβ3 or Kv1.5ΔC+Kvβ2 complexes did not respond to changes in intracellular pyridine nucleotide concentration, indicating that the C-terminus is required for pyridine nucleotide-dependent interactions between Kvβ and Kv1.5. A glutathione-S-transferase (GST) fusion protein containing the C-terminal peptide of Kv1.5 did not bind to apoKvβ2, but displayed higher affinity for Kvβ2:NADPH than Kvβ2:NADP+. The GST fusion protein also precipitated Kvβ proteins from mouse brain lysates. Pull-down experiments, structural analysis and electrophysiological data indicated that a specific region of the C-terminus (Arg543-Val583) is required for Kvβ binding. These results suggest that the C-terminal domain of Kv1.5 interacts with β-subunits and that this interaction is essential for the differential regulation of Kv currents by oxidized and reduced nucleotides.

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Abbreviations

Kvβ:

β-Subunit of the voltage-gated potassium channel

Kv:

Voltage-gated potassium channel

Kv∆C:

Deletion of the last C-terminal amino acids of Kv channel

GST:

Glutathione-S-transferase fusion protein

V h act:

Voltage at which half of the channels are activated

V h inact:

Voltage at which half of the channels are inactivated

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Acknowledgments

The authors thank Dr. Maiying Kong for her advice on statistical analysis, and Joshua Salabei and Ermin Villa for their help in cloning experiments. This work was partly supported by NIH grants RR024489 HL-55477, HL-59378 (to A.B.), HL-089372 (to O.A.B.), 0865466D American Heart Beginning-grant-in-aid, HL-102171, Deans Research Funding USF-COP (to S.M.T), and the Program of the Russian Academy of Sciences for the “Molecular and Cellular Biology” (to V.N.U).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, 33612, USA

    Srinivas M. Tipparaju

  2. Diabetes and Obesity Center, University of Louisville, Louisville, KY, 40202, USA

    Xiao-Ping Li, Peter J. Kilfoil, Aruni Bhatnagar & Oleg A. Barski

  3. Department of Molecular Medicine, University of South Florida, Tampa, FL, 33612, USA

    Bin Xue & Vladimir N. Uversky

  4. Institute for Biological Instrumentation, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russia

    Vladimir N. Uversky

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  1. Srinivas M. Tipparaju
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  2. Xiao-Ping Li
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Correspondence to Srinivas M. Tipparaju.

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Tipparaju, S.M., Li, XP., Kilfoil, P.J. et al. Interactions between the C-terminus of Kv1.5 and Kvβ regulate pyridine nucleotide-dependent changes in channel gating. Pflugers Arch - Eur J Physiol 463, 799–818 (2012). https://doi.org/10.1007/s00424-012-1093-z

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