Determinants of 4-Aminopyridine Sensitivity in a Human Brain Kv1.4 K+ Channel: Phenylalanine Substitutions in Leucine Heptad Repeat Region Stabilize Channel Closed State
- Susan I. V. Judge1,1,
- Jay Z. Yeh3,
- James E. Goolsby1,
- Mervyn J. Monteiro1,2 and
- Christopher T. Bever, Jr.1,1
- 1Research and Neurology Services, VA Maryland Health Care System, Baltimore, Maryland (S.I.V.J., C.T.B.); Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland (S.I.V.J., C.T.B., M.J.M., J.E G.); 2Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland (M.J.M.); and 3Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois (J.Z.Y.)
- Susan I. V. Judge, Ph.D. Department of Neurology, University of Maryland School of Medicine, BRB 12–040, 655 West Baltimore Street, Baltimore, MD 21201. E-mail:sjudge{at}umaryland.edu
Abstract
The biophysical and pharmacological effects of individual phenylalanine-for-leucine (Phe-for-Leu) substitutions in the leucine heptad repeat region located at the cytosolic surface of the channel pore, on whole-cell K+ currents, were studied in cloned and mutated human brain Kvl.4 K+ channels (hKvl.4) transiently transfected into HeLa cells. Although L2 and L5 are not considered part of the 4-aminopyridine (4-AP) binding site, unlike the L4 heptad leucine, Phe substitutions at L2 (L464) or L5 (L485) increase 4-AP sensitivity by 400-fold, as seen previously in the L4F mutant channel (Judge et al., 1999). Greater depolarizing shifts manifest in the voltage dependence of activation and inactivation in L2F (20 mV) and L5F (30 mV) than in L4F (10 mV) relative to hKv1.4. L1F (L457) and L3F (L471) increase 4-AP sensitivity by 8- and 150-fold, respectively, and produce depolarizing shifts in activation of ∼5 mV without affecting inactivation. The apparent free energy differences of 4-AP binding in each mutant suggest enhanced drug-channel interactions (L2F ≥ L4F ≥ L5F > L3F > L1F). Deactivation kinetics are accelerated in L2F (11-fold), L5F (8-fold), L1F (5-fold), and L3F (2-fold), at −50 mV. All Phe-for-heptad-Leu substitutions produce gating changes suggesting variable stabilization of the channel closed state conformation, with L1F, L2F, and L5F exhibiting the strongest correlations between altered gating and increased 4-AP sensitivity. If 4-AP blocks the open channel by promoting closure of the activation gate (recent Armstrong-Loboda model), then changes in the leucine heptad repeat that stabilize the channel closed state may contribute to increased 4-AP sensitivity by amplifying the mechanism of 4-AP block.
Footnotes
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This work was supported by separate Department of Veterans Affairs Merit Review Funding to S.I.V.J. and C.T.B. In addition, this work was supported by grant RG2127A2 from the National Multiple Sclerosis Society to C.T.B. and by grant AG11386 from the National Institutes of Health to M.J.M.
- Abbreviations:
- 4-AP
- 4-aminopyridine
- hKv1.4
- cloned human brain Kv1.4 potassium channel
- Sh
- Shaker
- SS-inactivation
- steady-state inactivation
- LB
- Luria broth
- τ
- time constant of inactivation
- τfast
- fast inactivation time constant
- τslow
- slow inactivation time constant
- τtails
- deactivation time constant
- V1/2
- midpoint potential
- ΔF
- apparent free energy difference of binding
- ρ
- correlation coefficient
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- Received September 14, 2001.
- Accepted January 15, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
