Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC308

Poster Communications

Protein Kinase A (PKA) is central for the forward transport of two-pore domain K+ channels K2P3.1 and K2P9.1

A. Mant1, D. Elliott2, P. A. Eyers3, I. M. O'Kelly1

1. Centre for Human Development, Stem Cells and Regeneration, University of Southampton, Southampton, United Kingdom. 2. Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom. 3. Yorkshire Cancer Research Institute for Cancer Studies, University of Sheffield, Sheffield, United Kingdom.

Acid sensitive two-pore domain K+ channels (K2P3.1 and K2P9.1) play key roles in physiology and disease, the most fundamental of which is control of the resting membrane potential of cells(1). As these ‘leak’ channels are constitutively active once expressed at the plasma membrane, tight control of surface expression is fundamental to the regulation of K+ flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the C-termini of K2P3.1 and K2P9.1 (S393 and S373, respectively) and overcomes retention in the endoplasmic reticulum by βCOP (2-4). We sought to identify the kinase responsible for phosphorylation of the terminal serine in human (h) and rat (r) K2P3.1 and K2P9.1. We tested the effect of mutating the terminal serine to alanine (S393A) on the function of hK2P3.1 expressed in Xenopus oocytes by two electrode voltage clamp. This mutation resulted in a total loss of current, as did the double substitution S392A/S393A or the removal of the terminal valine (ΔV394). Three candidate kinases were identified: cAMP-dependent protein kinase (PKA), ribosomal S6 kinase (RSK,) and protein kinase C (PKC). In vitro phosphorylation assays supported in silico predictions: PKA phosphorylated the terminal serine of both hK2P3.1 and h and rK2P9.1. RSK2 phosphorylated the terminal serine of hK2P3.1 effectively in vitro, but PKC did not appreciably phosphorylate any of the C-termini tested. Whole cell patch clamp measurements of hK2P3.1 expressed in HEK293 show a negative shift in resting membrane potential from -32.6 mV (S.E.M. 1.16, n=6) in non-transfected cells to 54.6 mV (S.E.M. 3.12, n=14) in channel-transfected cells. For cells transfected with hK2P3.1 and cultured in the presence of the constitutive PKA activator, 8Br-cAMP (0.4 mM), increased current was observed at test potentials between -50 mV and +90 mV. At 60 mV, evoked current increased from 0.59 pA (S.E.M 0.05, n=7) to 1.09 pA (S.E.M 0.14, n=9) in 8Br-cAMP treated cells, with a modest negative shift in the resting membrane potential to -58.6 mV (S.E.M. 2.45, n=7). Conversely, in HEK293 cells incubated with two different PKA inhibitors (1 µM KT5720 or 20 µM myristoylated PKA-specific inhibitor), there was a decrease in current at all test potentials when compared with non-treated cells (significant: P≤0.05). Unexpectedly, the RSK inhibitor, SL0101, caused an increase in current (1.01 pA, S.E.M 0.12 at 60 mV, n=8), but without a concomitant decrease in membrane potential compared to non-treated matched controls. Immunofluorescence and flow cytometric measurements of GFP-tagged K2P3.1 and K2P9.1 expressed in HEK293 cells supported the in vitro phosphorylation and electrophysiology conclusions: PKA is responsible for the phosphorylation of the terminal serine in both K2P3.1 and K2P9.1 (5).

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