Block of Cloned Voltage-Gated Potassium Channels by the Second Messenger
Diacylglycerol Independent of Protein Kinase C.
Bowlby, Mark R., and Irwin B. Levitan.
Department of Biochemistry and Center for Complex Systems, Brandeis
University, Waltham, MA 02254.
APStracts 2:0057N, 1995.
SUMMARY AND CONCLUSIONS
1. Diacylglycerols (DAG's) are common intracellular second messengers
produced as a result of activation of phospholipase C. We have examined the
direct effects of DAG on currents from cloned voltage dependent potassium
channels. Potassium channels were studied by overexpression of their cRNA's in
Xenopus oocytes or of their cDNA's in HEK 293 cells, and macroscopic currents
were recorded from inside-out membrane patches. 2. When applied to the
intracellular side of the patch, 1,2-dioctanoyl-sn-glycerol (C8:0) (DOG)
blocks Shaker IR, Kv1.3 and Kv1.6 channels. This block appears
macroscopically as a large speeding of the inactivation rate. Longer carbon
chain length DAG's (10 and 12 carbons) are less effective in producing this
response. 3. DOG is effective at low concentrations, doubling the apparent
inactivation rate at 162 nM, and has a fast time course, with a wash-in and
reversal to control each within about 30 s. 4. Voltage steps delivered with
a two pulse protocol in the presence of DOG indicate that recovery from DOG
block is voltage dependent. Recovery occurs quickly (_ = 507 ms) when channels
are closed quickly by hyperpolarized (-90 mV) potentials, and occurs slowly (_
= 1.3 s) when channels are closed incompletely by depolarized (-60 mV)
potentials. 5. The action of DOG is independent of protein kinase C (PKC)
activation, as it does not require ATP, nor is it blocked by staurosporin or
by the PKC inhibitor peptide 19-36. 6. DOG appears to block the open state
of these K + channels, rather than modulate gating, as DOG and the open
channel blocker tetraethylammonium (TEA) interact when blocking the Kv1.3
channel. The binding site for DOG, however, is not identical to that for TEA,
as the Shaker IR mutant T441S has a 10 fold lower affinity for TEA, yet its
affinity for DOG remains unchanged. 7. Diacylglycerols may prove to be
useful tools for probing the structure and function of voltage dependent K +
channels.
Received 13 December 1994; accepted in final form 14 February 1995.
APS Manuscript Number J774-4.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 3 April 1995.