Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Channels (Austin). May-Jun 2007;1(3):179-88. doi: 10.4161/chan.4437. Epub 2007 May 15.

Abstract

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Asparagine
  • Batrachotoxins / chemistry
  • Batrachotoxins / metabolism
  • Batrachotoxins / pharmacology*
  • Binding Sites
  • Cell Line
  • Cell Membrane Permeability
  • Humans
  • Ion Channel Gating / drug effects
  • Membrane Potentials
  • Models, Molecular
  • Molecular Structure
  • Muscle Proteins / antagonists & inhibitors*
  • Muscle Proteins / chemistry
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism
  • Mutation*
  • Myocytes, Cardiac / drug effects*
  • Myocytes, Cardiac / metabolism
  • NAV1.5 Voltage-Gated Sodium Channel
  • Protein Binding
  • Protein Conformation
  • Rats
  • Sodium / metabolism*
  • Sodium Channel Blockers / chemistry
  • Sodium Channel Blockers / metabolism
  • Sodium Channel Blockers / pharmacology*
  • Sodium Channels / chemistry
  • Sodium Channels / genetics
  • Sodium Channels / metabolism
  • Time Factors
  • Transfection

Substances

  • Batrachotoxins
  • Muscle Proteins
  • NAV1.5 Voltage-Gated Sodium Channel
  • SCN5A protein, human
  • Scn4a protein, rat
  • Scn5a protein, rat
  • Sodium Channel Blockers
  • Sodium Channels
  • Asparagine
  • Sodium