BLOCK OF CLONED VOLTAGE-GATED POTASSIUM CHANNELS BY THE 2ND MESSENGERDIACYLGLYCEROL INDEPENDENT OF PROTEIN-KINASE-C

1. Diacylglycerols (DAGs) are common intracellular second messengers p roduced as a result of activation of phospholipase C. We have examined the direct effects of DAG on currents from cloned voltage-dependent p otassium channels. Potassium channels were studied by overexpression o f their cRNAs in Xenopus oocytes or of their cDNAs in HEK 293 cells, a nd macroscopic currents were recorded from inside-out membrane patches . 2. When applied to the intracellular side of the patch, 1,2-dioctano yl-sn-glycerol (C8:0) (DOG) blocks Shaker IR, Kv1.3, and Kv1.6 channel s. This block appears macroscopically as a large speeding of the inact ivation rate. Longer carbon chain length DAGs (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 w ithin similar to 30 s. 4. Voltage steps delivered with a two pulse pro tocol in the presence of DOG indicate that recovery from DOG block is voltage dependent. Recovery occurs quickly (tau = 507 ms) when channel s are closed quickly by hyperpolarized (-90 mV) potentials, and occurs slowly (tau = 1.3 s) when channels are closed incompletely by depolar ized(-60 mV) potentials. 5. The action of DOG is independent of protei n kinase C (PKC) activation, because it does not require ATP, nor is i t blocked by staurosporin or by the PKC inhibitor peptide 19-36. 6. DO G appears to block the open state of these K+ channels, rather than mo dulate gating, because DOG and the open channel blocker tetraethylammo nium (TEA) interact when blocking the Kv1.3 channel. The binding site for DOG, however, is not identical to that for TEA, because the Shaker IR mutant T441S has a 10-fold lower affinity for TEA, yet its affinit y for DOG remains unchanged. 7. DAGs may prove to be useful tools for probing the structure and function of voltage-dependent K+ channels.