Potassium (K+) channels are critical determinants of the electrical activity of the heart and when they malfunction life-threatening arrhythmias can result. PIP2 is a signaling phospholipid that for the past two decades has been recognized as a master regulator of ion channel activity. Although we have learned the most in inwardly rectifying K (Kir) channels that control the resting potential of cells about how PIP2 causes + channel gating, our understanding of similar PIP2 control in voltage-gated K (Kv) channels that control the + repolarization phase of the action potential is lacking. This proposal aims to push our understanding to the next level in each of these two important classes of ion channels. For Kir channels, with a crystal structure of Kir3.2 in complex with PIP2 and our ability to produce full-length purified protein we are ready to probe the mechanism of control of activity by protein phosphorylation. For the past decade we have realized that Kir3 channel phosphorylation by specific kinases can stimulate (e.g. Protein Kinase A - PKA) or inhibit (e.g. Protein Kinase C - PKC) activity by enhancing or retarding sensitivity to PIP2, respectively. In this proposal we hypothesize that the strategic phosphorylation of specific residues by PKC compete with PIP2 for positively charged Kir3 channel-PIP2 interacting residues, thus weakening the channel's ability to coordinate PIP2 and causing inhibition of channel activity. We propose to test this hypothesis by identifying in vitro PKC phosphorylated residues of Kir3 channels using Mass Spectrometry and employing computational modeling, mutagenesis and electrophysiology to probe the mechanism by which phosphorylation of a specific residue alters channel-PIP2 interactions and inhibits channel activity. For Kv channels, we present strong preliminary results showing that PIP2 controls the Kv2.1 channel slow inactivation which like other (voltage-dependent and voltage-independent) channels leads to a collapse of the selectivity filter. These novel data lead us to hypothesize that Kv2.1 PIP2 interacting residues are linked to the selectivity filter via a relay residue in the middle of the pore-lining S6 helix. We propose to tes this hypothesis using computational models, mutagenesis and electrophysiology and establishing this novel mechanism for controlling Kv channel activity, which may operate in additional Kv channels that display slow inactivation. Results from the proposed studies in the 5 cycle of this grant will advance our understanding of PIP2 gating th and its modulation by post-translational modification mechanisms to adjust channel activity. Our structural insights from Kir3 channels will be beneficial in our identification of novel PIP2-sensitive Kv2.1 channel residues. Our novel insights of the coupling of Kv2.1 PIP2 interacting residues to the selectivity filter of this channel will certainly guide us to pursue long-term studies in Kir3 channels to compare the coupling of PIP2- interacting sites to the selectivity filter of these channels, a process that remains unclear in the Kir field.
This proposal utilizes state of the art technologies to investigate how the phospholipid PIP2, residing in the inner leaflet of the plasma membrane, controls the activity of two potassium channel proteins. Regulation of channel-PIP2 interactions and thus ion flow across the membrane underlies cardiac electrical activity.
| Corbin-Leftwich, Aaron; Small, Hannah E; Robinson, Helen H et al. (2018) A Xenopus oocyte model system to study action potentials. J Gen Physiol 150:1583-1593 |
| Ha, Junghoon; Xu, Yu; Kawano, Takeharu et al. (2018) Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). J Biol Chem 293:3546-3561 |
| Delgado-Ramírez, Mayra; De Jesús-Pérez, José J; Aréchiga-Figueroa, Iván A et al. (2018) Regulation of Kv2.1 channel inactivation by phosphatidylinositol 4,5-bisphosphate. Sci Rep 8:1769 |
| Fribourg, Miguel; Logothetis, Diomedes E; González-Maeso, Javier et al. (2017) Elucidation of molecular kinetic schemes from macroscopic traces using system identification. PLoS Comput Biol 13:e1005376 |
| Tobelaim, William S; Dvir, Meidan; Lebel, Guy et al. (2017) Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels. Channels (Austin) 11:686-695 |
| Tobelaim, William Sam; Dvir, Meidan; Lebel, Guy et al. (2017) Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel. Proc Natl Acad Sci U S A 114:E869-E878 |
| Younkin, Jason; Gaitonde, Supriya A; Ellaithy, Amr et al. (2016) Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists. ACS Chem Neurosci 7:1292-9 |
| Moreno, José L; Miranda-Azpiazu, Patricia; García-Bea, Aintzane et al. (2016) Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia. Sci Signal 9:ra5 |
| Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie et al. (2016) Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms. Cell Rep 14:129-139 |
| Li, Junwei; Xiao, Shaoying; Xie, Xiaoxiao et al. (2016) Three pairs of weak interactions precisely regulate the G-loop gate of Kir2.1 channel. Proteins 84:1929-1937 |
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