Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C13
Phospholipid activation of M-type potassium channels
V. S. Telezhkin1, A. Gibb1, D. A. Brown1
1. Neurosciences, Physiology & Pharmacology, University College London, London, United Kingdom.
The primary membrane phospholipid involved in regulating M-type (Kv7.2/7.3) potassium channels is likely to be inositol-4,5-bisphosphate (PI(4,5)P2). Channels open on binding of PI(4,5)P2, while dissociation of PI(4,5)P2 [1,2], which may be caused by depletion of membrane PI(4,5)P2 following (e.g.) stimulation of muscarinic acetylcholine receptors, results in closure of Kv7.2/7.3 channels . Although stimulation of Kv7.2/7.3 channels by PI(4,5)P2 is well established, the contribution of other membrane phospholipids has not been widely studied, but it is known that the homologous Kv7.1 protein can bind a range of anionic phospholipids . The aim of this study was to compare activation of Kv7.2/7.3 channels stably expressed in Chinese Hamster Ovary cells by dioctanoyl-(DiC8)-phosphoinositides and sphingosine phosphates applied in incremental concentrations via a fast microperfusion technique to isolated inside-out membrane patches held at -15 mV . PI(4,5)P2 is an aliphatic compound which consists of a negatively charged hydrophilic inositol-1,4,5-trisphosphate (I(1,4,5)P3) head-group and a neutral hydrophobic diacylgycerol tail. PI(4)P, the precursor of PI(4,5)P2 and the product of its further phosphorylation, PI(3,4,5)P3, share conceptually similar structures. The effects of these phosphoinositides as well as I(1,4,5)P3 and I(4,5)P2 were compared with the effects of DiC8-PI(4,5)P2. The concentration-dependence of Kv7.2/7.3 channel activation was characterised by two component curves . Different phosphoinositides yielded similar Km values for the ‘high-affinity’ component but different ‘low-affinity’ Km2 values (DiC8-PI(3,4,5)P3 32.8 ± 2.5 μM (n = 15); DiC8-PI(4,5)P2 72.5 ± 5.4 μM (n = 33); DiC8-PI(4)P 83.5 ± 11.6 μM (n = 18)). The water soluble inositol phosphates I(1,4,5)P3 and I(4,5)P2 caused no observable channel activation. Therefore it seems that stimulation of Kv7.2/7.3 channels requires at least two necessary factors: a negative phosphate group, and a hydrophobic moiety for an appropriate orientation in the membrane. We tested this further using sphingosine-1-phosphate (S1P), a membrane phospholipid whose aliphatic structure corresponds in principle to PI(4,5)P2. Being highly hydrophobic, S1P was dissolved in methanol, which is known to inhibit potassium channels; even so, S1P effectively stimulated Kv7.2/7.3 channels. In conclusion, these data suggest that the aliphatic nature of PI(4,5)P2 holds the key to Kv7.2/7.3 channel activation, with minor discrimination between different phosphates.
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