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NEUROPHARMACOLOGY

4-Aminopyridine Prevents the Conformational Changes Associated with P/C-Type Inactivation in Shaker Channels

Departments of Anesthesiology, Pharmacology & Therapeutics (T.W.C., D.F.) and Cellular and Physiological Sciences (M.V., S.R., S.J.K.), University of British Columbia, Vancouver, British Columbia, Canada

The effect of 4-aminopyridine (4-AP) on K_v channel activation has been extensively investigated, but its interaction with inactivation is less well understood. Voltage-clamp fluorimetry was used to directly monitor the action of 4-AP on conformational changes associated with slow inactivation of Shaker channels. Tetramethylrhodamine-5-maleimide was used to fluorescently label substituted cysteine residues in the S3-S4 linker (A359C) and pore (S424C). Activation- and inactivation-induced changes in fluorophore microenvironment produced fast and slow phases of fluorescence that were modified by 4-AP. In Shaker A359C, 4-AP block reduced the slow-phase contribution from 61 ± 3 to 28 ± 5%, suggesting that binding inhibits the conformational changes associated with slow inactivation and increased the fast phase that reports channel activation from 39 ± 3 to 72 ± 5%. In addition, 4-AP enhanced both fast and slow phases of fluorescence return upon repolarization ( reduced from 87 ± 15 to 40 ± 1 ms and from 739 ± 83 to 291 ± 21 ms, respectively), suggesting that deactivation and recovery from inactivation were enhanced. In addition, the effect of 4-AP on the slow phase of fluorescence was dramatically reduced in channels with either reduced (T449V) or permanent P-type (W434F) inactivation. Interestingly, the slow phase of fluorescence return of W434F channels was enhanced by 4-AP, suggesting that 4-AP prevents the transition to C-type inactivation in these channels. These data directly demonstrate that 4-AP prevents slow inactivation of K_v channels and that 4-AP can bind to P-type-inactivated channels and selectively inhibit the onset of C-type inactivation.

Address correspondence to: Dr. David Fedida, Department of Anesthesiology, Pharmacology and Therapeutics and Cellular and Physiological Sciences, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. E-mail: fedida{at}interchange.ubc.ca

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				M. Vaid, T. W. Claydon, S. Rezazadeh, and D. Fedida Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel J. Gen. Physiol., July 28, 2008; 132(2): 209 - 222. [Abstract] [Full Text] [PDF]