AJP - Cell Physiology, Vol 263, Issue 5 C1081-C1087, Copyright © 1992 by American Physiological Society
Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells
A. M. Frace and D. C. Eaton
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
The effects of amino group specific reagents were examined on single,
large-conductance, Ca(2+)-activated, K+ channels in excised membrane
patches from GH3 cells. The reagents used include trinitrobenzene sulfonic
acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido
derivative, and sulfophenyl-isothiocyanate. These reagents react covalently
with peptide terminal amino groups and the epsilon amino groups of lysine
residues, thereby removing positive charge. Internal application of 0.1-1.0
mM reagent to inside-out patches irreversibly increases channel open
probability. Single-channel conductance and voltage sensitivity are not
affected by modification. Analysis of channel openings and closures shows
that the increase in open probability is predominantly due to the loss of
long-duration closures of the channel; however, the lengths of
long-duration openings are increased. After the modification in the
presence of Ca2+ was performed, the channel open probability remains large,
regardless of the internal Ca2+ concentration. Transitions among several
open and closed states of the modified channel are present in the absence
of Ca2+, suggesting that many state transitions are not directly dependent
on Ca2+ binding or dissociation.
