Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle.
We have studied potassium currents through a cloned Ca(2+)-dependent K+ channel (hslo) from human myometrium. Currents were recorded in inside-out macropatches from membranes of Xenopus laevis oocytes. In particular, the inactivation-like process that these channels show at high positive potentials was assessed in order to explore its molecular nature. This current inhibition conferred a bell shape to the current-voltage curves. The kinetic and voltage dependence of this process suggested the possibility of a Ba2+ block. There were the following similarities between the inactivation process observed at zero-added Ba2+ and the internal Ba2+ block of hslo channels: (a) in the steady state, the voltage dependence of the current inhibition observed at zero-added Ba2+ was the same as the voltage dependence of the Ba2+ block; (b) the time constant for recovery from current decay at zero-added Ba2+ was the same as the time constant for current recovery from Ba2+ blockade; and (c) current decay was largely suppressed in both cases by adding a Ba2+ chelator [(+)-18-crown-6-tetracarboxylic acid] to the internal solution. In our experimental conditions, we determined that the Kd for the complex chelator-Ba2+ is 1.6 x 10(-10) M. We conclude that the current decay observed at zero-added Ba2+ to the internal solution is due to contaminant Ba2+ present in our solutions (approximately 70 nM) and not to an intrinsic gating process. The Ba2+ blocking reaction in hslo channels is bimolecular. Ba2+ binds to a site (Kd = 0.36 +/- 0.05 mM at zero applied voltage) that senses 92 +/- 25% of the potential drop from the internal membrane surface.
Diaz
F
F
Centro de Estudios Cientificos de Santiago, Universidad de Chile.
Wallner
M
M
Stefani
E
E
Toro
L
L
Latorre
R
R
D000818:Animals; D001464:Barium / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004594:Electrophysiology; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D002614:Chelating Agents; D004988:Ethers, Cyclic; D008051:Lipid Bilayers; D015221:Potassium Channels; D001464:Barium; D002118:Calcium