Native Kv1.3 channels are upregulated by protein kinase C.
The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation-through effects on membrane potential, Ca2+ signalling, and interleukin-2 secretion-and in cytotoxic killing and volume regulation. Because PKC activation (as well as increased intracellular Ca2+) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by +8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action with "death brew" (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K+ conductance (GK) by 41 +/- 2%. PKC activation by 4 beta-phorbol 12,13-dibutyrate, increased GK by 69 +/- 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate or substrate peptides reduced GK by 43 +/- 5% and 38 +/- 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose- and light-dependent manner (IC50 approximately 250 nM). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca2+ entry in the presence of activating stimuli.
Chung
I
I
Department of Physiology, University of Toronto, Ontario, Canada.
Schlichter
L C
LC
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport; D051664:Kv1.3 Potassium Channel; D008564:Membrane Potentials; D009281:Naphthalenes / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D015221:Potassium Channels / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D015854:Up-Regulation
D004791:Enzyme Inhibitors; C496973:KCNA3 protein, human; D051664:Kv1.3 Potassium Channel; D009281:Naphthalenes; D010703:Phorbol Esters; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; C058819:calphostin C; D002118:Calcium