Diomedes E. Logothetis

PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain

The subjective feeling of cold is mediated by the activation of TRPM8 channels in thermoreceptive neurons by cold or by cooling agents such as menthol. Here, we demonstrate a central role for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the activation of recombinant TRPM8 channels by both cold and menthol. Moreover, we show that Ca2+ influx through these channels activates a Ca2+-sensitive phospholipase C and that the subsequent depletion of PI(4,5)P2 limits channel activity, serving as a unique mechanism for desensitization of TRPM8 channels. Finally, we find that mutation of conserved positive residues in the highly conserved proximal C-terminal TRP domain of TRPM8 and two other family members, TRPM5 and TRPV5, reduces the sensitivity of the channels for PI(4,5)P2 and increases inhibition by PI(4,5)P2 depletion. These data suggest that the TRP domain of these channels may serve as a PI(4,5)P2-interacting site and that regulation by PI(4,5)P2 is a common feature of members of the TRP channel family.

PIP2 Activates KCNQ Channels, and Its Hydrolysis Underlies Receptor-Mediated Inhibition of M Currents

KCNQ channels belong to a family of potassium ion channels with crucial roles in physiology and disease. Heteromers of KCNQ2/3 subunits constitute the neuronal M channels. Inhibition of M currents, by pathways that stimulate phospholipase C activity, controls excitability throughout the nervous system. Here we show that a common feature of all KCNQ channels is their activation by the signaling membrane phospholipid phosphatidylinositol-bis-phosphate (PIP(2)). We show that wortmannin, at concentrations that prevent recovery from receptor-mediated inhibition of M currents, blocks PIP(2) replenishment to the cell surface. Moreover, we identify a C-terminal histidine residue, immediately proximal to the plasma membrane, mutation of which renders M channels less sensitive to PIP(2) and more sensitive to receptor-mediated inhibition. Finally, native or recombinant channels inhibited by muscarinic agonists can be activated by PIP(2). Our data strongly suggest that PIP(2) acts as a membrane-diffusible second messenger to regulate directly the activity of KCNQ currents.

Alterations in conserved Kir channel-PIP2 interactions underlie channelopathies.

Inwardly rectifying K(+) (Kir) channels are important regulators of resting membrane potential and cell excitability. The activity of Kir channels is critically dependent on the integrity of channel interactions with phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we identify and characterize channel-PIP(2) interactions that are conserved among Kir family members. We find basic residues that interact with PIP(2), two of which have been associated with Andersens and Bartters syndromes. We show that several naturally occurring mutants decrease channel-PIP(2) interactions, leading to disease.

Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.

Direct interactions of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) with inwardly rectifying potassium channels are stronger with channels rendered constitutively active by binding to PtdIns(4,5)P2, such as IRK1, than with G-protein-gated channels (GIRKs). As a result, PtdIns(4,5)P2 alone can activate IRK1 but not GIRKs, which require extra gating molecules such as the βγ subunits of G proteins or sodium ions. Here we identify two conserved residues near the inner-membrane interface of these channels that are critical in interactions with PtdIns(4,5)P2. Between these two arginines, a conservative change of isoleucine residue 229 in GIRK4 to the corresponding leucine found in IRK1 strengthens GIRK4–PtdIns(4,5)P2 interactions, eliminating the need for extra gating molecules. A negatively charged GIRK4 residue, two positions away from the most strongly interacting arginine, mediates stimulation of channel activity by sodium by strengthening channel–PtdIns(4,5)P2 interactions. Our results provide a mechanistic framework for understanding how distinct gating mechanisms of inwardly rectifying potassium channels allow these channels to subserve their physiological roles.

Receptor-mediated hydrolysis of plasma membrane messenger PIP 2 leads to K + -current desensitization

Phosphatidylinositol bisphosphate (PIP2) directly regulates functions as diverse as the organization of the cytoskeleton, vesicular transport and ion channel activity. It is not known, however, whether dynamic changes in PIP2 levels have a regulatory role of physiological importance in such functions. Here, we show in both native cardiac cells and heterologous expression systems that receptor-regulated PIP2 hydrolysis results in desensitization of a GTP-binding protein-stimulated potassium current. Two receptor-regulated pathways in the plasma membrane cross-talk at the level of these channels to modulate potassium currents. One pathway signals through the βγ subunits of G proteins, which bind directly to the channel. Gβγ subunits stabilize interactions with PIP2 and lead to persistent channel activation. The second pathway activates phospholipase C (PLC) which hydrolyses PIP2 and limits Gβγ-stimulated activity. Our results provide evidence that PIP2 itself is a receptor-regulated second messenger, downregulation of which accounts for a new form of desensitization.

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides.

Activation of several inwardly rectifying K+ channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P2 strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P2. We investigated whether these inwardly rectifying K+ channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. In contrast, IRK1 channels were not activated by PtdIns(3,4)P2 and only marginally by high concentrations of PtdIns(3,5)P2. Similarly, high concentrations of PtdIns(3,4,5)P3 were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P2, whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P2 and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P2. Incorporation of PtdInsP2 into the membrane is necessary for activation, as the short chain water soluble diC4 PtdIns(4,5)P2 did not activate either channel, whereas activation by diC8 PtdIns(4, 5)P2 required high concentrations.

Characteristic Interactions with Phosphatidylinositol 4,5-Bisphosphate Determine Regulation of Kir Channels by Diverse Modulators

The activity of specific inwardly rectifying potassium (Kir) channels is regulated by any of a number of different modulators, such as protein kinase C, Gq -coupled receptor stimulation, pH, intracellular Mg2+ or the βγ-subunits of G proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an essential factor for maintenance of the activity of all Kir channels. Here, we demonstrate that the strength of channel-PIP2 interactions determines the sensitivity of Kir channels to regulation by the various modulators. Furthermore, our results suggest that differences among Kir channels in their specific regulation by a given modulator may reflect differences in their apparent affinity of interactions with PIP2.

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Phosphoinositides are critical regulators of ion channel and transporter activity. Defects in interactions of inwardly rectifying potassium (Kir) channels with phosphoinositides lead to disease. ATP-sensitive K+ channels (KATP) are unique among Kir channels in that they serve as metabolic sensors, inhibited by ATP while stimulated by long-chain (LC) acyl-CoA. Here we show that KATP are the least specific Kir channels in their activation by phosphoinositides and we demonstrate that LC acyl-CoA activation of these channels depends on their low phosphoinositide specificity. We provide a systematic characterization of phosphoinositide specificity of the entire Kir channel family expressed in Xenopus oocytes and identify molecular determinants of such specificity. We show that mutations in the Kir2.1 channel decreasing phosphoinositide specificity allow activation by LC acyl-CoA. Our data demonstrate that differences in phosphoinositide specificity determine the modulation of Kir channel activity by distinct regulatory lipids.

N-terminal transmembrane domain of the SUR controls trafficking and gating of Kir6 channel subunits

The sulfonylurea receptor (SUR), an ATP‐binding cassette (ABC) protein, assembles with a potassium channel subunit (Kir6) to form the ATP‐sensitive potassium channel (KATP) complex. Although SUR is an important regulator of Kir6, the specific SUR domain that associates with Kir6 is still unknown. All functional ABC proteins contain two transmembrane domains but some, including SUR and MRP1 (multidrug resistance protein 1), contain an extra N‐terminal transmembrane domain called TMD0. The functions of any TMD0s are largely unclear. Using Xenopus oocytes to coexpress truncated SUR constructs with Kir6, we demonstrated by immunoprecipitation, single‐oocyte chemiluminescence and electrophysiological measurements that the TMD0 of SUR1 strongly associated with Kir6.2 and modulated its trafficking and gating. Two TMD0 mutations, A116P and V187D, previously correlated with persistent hyperinsulinemic hypoglycemia of infancy, were found to disrupt the association between TMD0 and Kir6.2. These results underscore the importance of TMD0 in KATP channel function, explaining how specific mutations within this domain result in disease, and suggest how an ABC protein has evolved to regulate a potassium channel.

PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain K+ channels.

Two‐pore (2‐P) domain potassium channels are implicated in the control of the resting membrane potential, hormonal secretion, and the amplitude, frequency and duration of the action potential. These channels are strongly regulated by hormones and neurotransmitters. Little is known, however, about the mechanism underlying their regulation. Here we show that phosphatidylinositol 4,5‐bisphosphate (PIP2) gating underlies several aspects of 2‐P channel regulation. Our results demonstrate that all four 2‐P channels tested, TASK1, TASK3, TREK1 and TRAAK are activated by PIP2. We show that mechanical stimulation may promote PIP2 activation of TRAAK channels. For TREK1, TASK1 and TASK3 channels, PIP2 hydrolysis underlies inhibition by several agonists. The kinetics of inhibition by the PIP2 scavenger polylysine, and the inhibition by the phosphatidylinositol 4‐kinase inhibitor wortmannin correlated with the level of agonist‐induced inhibition. This finding suggests that the strength of channel PIP2 interactions determines the extent of PLC‐induced inhibition. Finally, we show that PIP2 hydrolysis modulates voltage dependence of TREK1 channels and the unrelated voltage‐dependent KCNQ1 channels. Our results suggest that PIP2 is a common gating molecule for K+ channel families despite their distinct structures and physiological properties.