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Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
We used
single-channel recording techniques to identify and characterize a
large-conductance,
Ca2+-independent
K+ channel in the colonic
secretory cell line T84. In symmetric potassium gluconate, this channel
had a linear current-voltage relationship with a single-channel
conductance of 161 pS. Channel open probability
(Po) was
increased at depolarizing potentials. Partial substitution of bath
K+ with
Na+ indicated a permeability ratio
of K+ to
Na+ of 25:1. Channel
Po was reduced by
extracellular Ba2+. Event-duration
analysis suggested a linear kinetic model for channel gating having a
single open state and three closed states: C3
C2C1O.
Arachidonic acid (AA) increased the
Po of the
channel, with an apparent stimulatory constant
(Ks)
of 1.39 µM. Neither channel open time (O) nor the fast closed time
(C1) was affected by AA. In
contrast, AA dramatically reduced mean closed time by decreasing both
C3 and
C2. The
cis-unsaturated fatty acid linoleate increased Po
also, whereas the saturated fatty acid myristate and the
trans-unsaturated fatty acid elaidate
did not affect
Po. This channel
is activated also by negative pressure applied to the pipette during
inside-out recording. Thus we determined the effect of the
stretch-activated channel blockers amiloride and Gd3+ on the
K+ channel after activation by AA.
Amiloride (2 mM) on the extracellular side reduced single-channel
amplitude in a voltage-dependent manner, whereas
Gd3+ (100 µM) had no effect on
channel activity. Activation of this K+ channel may be important during
stimulation of Cl
secretion
by agonists that use AA as a second messenger (e.g., vasoactive
intestinal polypeptide, adenosine) or during the volume regulatory
response to cell swelling.
potassium channel; chloride secretion; fatty acids; intestine
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