Diomedes E. Logothetis - US grants

All cloned voltage-dependent ion channels show a striking conservation of regularly spaced, positively charged amino acids in the putative transmembrane domain, termed S4. It has been suggested that the S4 is the voltage sensor. The broad, long-term objectives of this proposal are to evaluate the role of the S4 region in voltage sensing. We have cloned K channels from muscle (RMK1) and brain (RCK1) that when expressed in oocytes or cells show properties of a delayed rectifier K channel (1). Specific mutations of three S4 charged residues showed that the charges R1 and R2 participate in the charge movement while K7 does not. Yet, each one of these charges makes quantitatively very different contributions to the overall gating valence (z). R2 accounts for 20% of z and shows a purely electrostatic behavior. R1 accounts for 40% of z, an effect that cannot be explained by electrostatic interactions alone (2). Yet, the relative contribution of residues R3, R4, K5, and R6 remains unknown since specific mutations of these residues have resulted in complete loss of function. The specific aims of this proposal are: 1) Having first identified and localized the RMK1 channel proteins (in particular the non-functional mutants) by immunofluorescence and immunoprecipitation we will embark on direct measurements of the gating charge movement by measuring gating currents; 2) We will assess the relative contribution of each K channel subunit S4 region in voltage sensing by creating tandem RMK1 constructs; 3) Effects of the specific amino acid content of S4 in voltage sensing by transplanting S4 regions from other channels into RMK1 and comparing the effects in the voltage sensing ability of the engineered molecules to those of the control donor ones; 4) Identification of acidic residues in putative transmembrane domains that may form salt links with the basic S4 residues. The experimental design and the methods used to carry out these projects include: site-directed mutagenesis; in vitro transcription and injection of mRNA into Xenopus oocytes; functional assessment by two microelectrode voltage clamping of oocytes of patch clamping of oocytes and cells. The RMK1 channel (each mutant separately) will be transfected into temperature sensitive (ts) COS cells which allow high level expression and, thus, direct measurements of gating charge movement with gating currents. The delayed rectifier channel offers a distinct advantage over rapidly inactivating currents by providing steady state measurements of current activation. Activation is very rapid within a small range of membrane potentials. The proposed experiments represent a comprehensive study to investigate the structural basis of voltage sensing. A better understanding of this phenomenon is essential given the central role that voltage gated ion channels play in excitable cell function.

This proposal seeks to examine in detail the nature of GTP-binding (G) protein activation of the muscarinic potassium (GIRK or KGIRI) channel. It is thought that agonist binding to its transmembrane receptor (R) causes dissociation of the heterotrimeric G protein into its component subunits (Galpha and Gbetagamma) which in turn activate KGIRI channels. Despite intense studies of this system in native membranes, major questions remain. What is the physiological role of differential activation of the channel by one the other or both G-protein subunit components? The answer to this question has not yet been addressed in any system which shows differential regulation by specific G-protein subunits. Cloning of KGIRI channel isoforms has made it feasible to use recombinant components of this system to identify unequivocally the specific effector G-protein subunits as well as the sites in the channel that they act upon. Use of synthetic peptides which affect activation of the channel by specific G-protein subunits can serve as a useful tool in distinguishing physiologically relevant G-protein subunit regulation of the channel. Moreover, structural information derived from such peptides can be used as probes to identify the channel sites of action of specific G-protein subunits. We propose the following two specific aims; (1) Effects of G- protein subunits on native and recombinant KGIRI channels. Recombinant G- protein subunits and KGIRI channels will be coexpressed in Xenopus oocytes and mammalian cell lines. Coexpression of specific G-protein subunits (Gbetagamma or mutant-activated Galpha*) with KGIRI isoforms will test for constitutive activation of these channels. Differential response of KGIRI clones and isoforms to G-protein subunits will be related to their primary structures. Finally, peptides, like QEHA and Galphai 297- 318, will be used as tools to distinguish between activation of Galpha and Gbetagamma subunits in membranes containing native (atrial) or recombinant (oocytes, CHO cells) components of the system. (2) Identification of KGIRI channel regions and amino acid residues responsible for activation by G-protein subunits. The second aim will attempt to identify the regions of KGIRI channels responsible for the activation by G-protein subunits. Crosslinking experiments with peptides shown to affect channel activation by specific G-protein subunits will help localize the relevant regions of interaction. A chimeric approach between two highly related K channels (KGIRIalpha and hpK ATPI), which maybe differentially affected by G-protein subunits, will also be used towards identification of regions responsible for interactions with Gbetagamma or Galpha* subunits. Once the important region(s) have been identified, site-directed mutagenesis will follow to identify the critical amino acid residues of the channel responsible for G- protein subunit activation. Specific amino acid substitutions suggested by the QEHA peptide results will be attempted.

Molecular studies of ion channel function have primarily focused on the proteins themselves or on their interactions with other proteins or ions. G protein-gated K channels (KG) for example are thought to be directly activated by the betagamma subunits of GTP binding proteins (Gbetagamma subunits). Current models of KG channel activation involve G protein subunit separation (which renders them active) and interaction with the channel subunits. Our preliminary results suggest that the Gbetagamma subunit/KG channel interaction requires the presence of PIP2 in the membrane in order to manifest its effects on channel activity. This surprising result is accompanied by other effects directly attributable to PIP2, such as the MgATP-dependent sensitization of KG channels to gating by internal Na ions and possibly the MgATP-dependent rundown of G protein stimulation of KG channel activity. These results together with two recent reports on the related inwardly rectifying channel KATP and on the Na / Ca transporter (but not on Na channels or Na / K pumps) herald the potential of an unexplored area of research, crucial to the functional integrity of membrane proteins. Our proposal aims to study in detail the effects of lipids, and in particular phospholipids on KG channel function. The experiments outlined will test further the molecular basis and significance of the PIP2 effects on KG channel activity and the dependence of G protein subunit KG channel activation on the presence of PIP2. It has been proposed that the lipid effects are electrostatic in nature. We will test this hypothesis and seek to identify the basic residues in the channel sequence constituting sites of interaction with the anionic phospholipids. Phospholipids of the phosphoinositide cycle allow dynamic participation of lipids in signaling. We believe that a better appreciation of the molecular details of KG channel function afforded by this study will allow more successful manipulation of this atrial channel in the control of supraventricular arrhythmias. For example, our recent discovery of the MgATP-dependent sensitization of the atrial KG channel (KACh) to gating by internal Na ions allowed us to demonstrate that digitalis treatment causes atrial cells to activate KACh (due to the Na accumulation it causes), providing an important link to the long known effects of this drug on supraventricular rhythm.

The G protein-gated inwardly rectifying K+ (GIRK) channel was the first example of a protein whose function was shown to be regulated by direct interactions with the betagamma subunits of GTP-binding (G) proteins (Logothetis et al., 1987). In this FIRCA they proposed to identify the region(s) in Gbeta subunits that interact(s) with GIRK channels. While Gbeta1-beta4 can activate GIRK channels with similar efficiency, Gbeta5 fails to do so even though it shows intact expression. They will use a chimeric strategy between Gbeta1 and Gbeta5 to identify the region between the two isoforms that is responsible for the difference in their abilities to stimulate GIRK activity. As they have already narrowed the region down to 95 amino acids, they will further narrow down this region utilizing a similar chimeric approach, so that through site-directed mutagenesis they can identify the specific amino acid residues responsible for the functional differences between the two beta subunits. Using a deletion mutagenesis approach they will attempt to identify the minimal Gbeta regions capable of binding the channel. Design of Gbeta peptides capable of affecting channel activity will follow identification of the minimal Gbeta regions.

DESCRIPTION (the applicant's description verbatim): Heterotrimeric GTP-binding (G) proteins transduce external signals that act on G Protein Coupled Receptors (GPCRs). When a signal binds its GPCR, it is thought that the activated G protein splits into its component subunits, the alpha-GTP (Ga-GTP) subunit and the betagamma (Gbg) complex, thus generating two signaling molecules that interact with specific effectors. Ga effector targets have long been recognized and studied extensively. The effector function of the bg complex was recognized more recently and although controversial at first, it is now well accepted. Gbg targets include, ion channels, (e.g. K+ (or GIRK) channels, neuronal type Ca2+ channels and the tetrodotoxin-insensitive Na+ channels), enzymes (e.g. PLA2, PLCb isoforms, adenylyl cyclase isoforms) and kinases (e.g. PI3K, the Bruton and Tsk tyrosine kinases). The list of effector targets continues to grow for each of the two signaling components of G proteins. Multiple isoforms for each of the subunits (20 Ga, 5 Gb and 14 Gg) have been identified. In vitro or heterotogous expression experiments have shown that unlike Ga subunit isoforms (4Gas, 3 Gai, 2 Gao, 4Gaq/11, etc.) which seem to associate with specific GPCRs and specific effectors, most Gbg combinations show little to no specificity towards their effector partners. Yet, in vivo (meaning in native tissues), there is exquisite specificity of Gbg signaling, so that for example the Gbg complexes associated with Gaq/11 do not signal to GIRK channels but those associated with Gai/o subunits do. The mechanism responsible for specificity of Gbg action is poorly understood, although it is basic to our understanding of G protein signaling at large. In this competitive renewal grant, we propose experiments in heterologous expression systems geared to understand the molecular basis for the specificity of Gbg signaling. In our ending current award we have identified functionally important amino acid residues on GIRK channels and on the Gbg subunits. We propose to identify residues in each protein that interact with the other, using a powerful mutant screening assay in yeast. In heterologous expression systems, where we have reconstituted specific Gbg signaling, we will determine the particular G protein subunits involved in the signaling. Ga subunits associated directly with the Gbg subunits seem to affect Gb's interactions with the K+ channel. We propose studies to elucidate the molecular basis for the dependence of the effector function of Gbg on Ga. We will test the hypothesis that distinct Ga subunits may determine specificity of Gbg signaling, using electrophysiology, molecular biology, biochemistry and fluorescence spectroscopy.

DESCRIPTION (provided by applicant): In this competitive renewal grant, we propose to continue moving forward our research program toward our long-term objective to understand at a molecular level how the signaling phospholipid PIP2 controls the activity of diverse ion channels. In the last cycle of this research program, based on available crystal structures, we developed 3-D models of Kir channels, such as Kir2.1, docked 4 PIP2 molecules onto the model , and guided by our mutagenesis data, chose the size of the system to perform Molecular Dynamics (MD) simulations. Such simulations predicted mostly interacting residues that had been previously implicated in PIP2 sensitivity by mutagenesis work and additional residues that still need to be tested experimentally. Our simulations revealed the Na coordination site in the CD-loop of Kir channels and guided us to identify a similar coordination site in the diverse Slo2.2 channel. Moreover, comparing simulations with and without PIP2 revealed explicit changes in networks of interactions that stabilize either the closed (in the absence of PIP2) or open (in the presence of PIP2) state. We have experimentally probed one such network by mutagenesis of specific interactions and the results support strongly the predictions of our theoretical model. We propose to perform a comprehensive study of key changes caused by the presence of PIP2 in the mammalian Kir2.2 channel, whose structure was recently solved. MD simulations of Kir2.2 with and without PIP2 reveal key roles for stabilization of two of the three channel gates in the closed or open states: the slide helix, B-loop and CD-loop stabilizes the G-loop gate; while the pore helix, and the two transmembrane domains stabilize the selectivity filter gate. Mutagenesis analysis will explicitly test these predictions and guide further simulations to elucidate the role of specific mutants. In parallel, as part of the current grant we have shown experimentally that PIP2 stabilizes the closed state of the gate controlled by the voltage sensor, while it also stabilizes an open state of another yet non- identified gate. Borrowing from what we have learned from Kir channels, we propose a similar analysis on Kv1.2, where MD simulations with and without PIP2 will guide experiments to elucidate how PIP2 controls Kv channel gates in this complex way. Relevance Pursuing the molecular mechanism by which PIP2 controls channel gating and voltage sensitivity, using two recently determined ion channel structures, will further our biophysical understanding of three diverse channels and potentially help with drug design for cardiac arrhythmias.

[unreadable] DESCRIPTION (provided by applicant) [unreadable] It has been recently appreciated that a common characteristic of members of the inwardly rectifying K+ (Kir) channel family is that they are all activated by phospatidylinositol-bis-phosphate (PIP2). Differences in channel-PIP2 interactions have been described among specific Kir members, both biochemically (Huang et al., 1998) and functionally (Huang et al., 1998; Zhang et al., 1999; Lopes et al., 2002). There seem to be Kir channels that show either relatively strong, intermediate or weak interactions with PIP2. Certain Kir channels are inhibited by PIP2 hydrolysis. The strength of channeI-PIP2 interactions correlates with the extent of channel activity and the degree of channel inhibition caused by signals that lead to PIP2 hydrolysis. Thus, channels that interact weakly with PIP2 are inhibited the most by PIP2 hydrolysis, while channels that interact strongly with PIP2 are not inhibited by PIP2 hydrolysis. Channel modulation by PKC affects channel activity in a manner dependent on channel-PIP2 interactions. Thus, in a wild-type channel showing PKC-mediated inhibition of activity, mutations strengthening channel-PIP2 interactions can attenuate or abolish the effect of PKC. Similarly, in a channel that interacts strongly with PIP2 and therefore lacks PKC-dependent inhibition, mutations that weaken channeI-PIP2 interactions can render the channel inhibitable by activated PKC. The PKC-mediated inhibition could be obtained in heterologous systems such as in Xenopus oocytes but not in others, such as the CHO mammalian cells. The current proposal aims to find answers to the following two major questions. (a) Are there specific PKC isoforms that are needed to reconstitute the PKC-mediated current inhibition in certain cell systems where the effect is absent? Are there specific PKC adaptor proteins involved in mediating the PKC effects? (b) To what extend is the muscarinic induced inhibition of Kir currents due to PKC mediated effects? Does PKC modulation of Kir channel activity weaken channel-PIP2 interactions? The current proposal expands and enhances the parent grant HL59949. In the parent grant our emphasis is to identify the PKC-dependent phosphorylation sites either on the channel protein itself or associated proteins and to study their relationship to channeI-PIPz interacting sites. In the current grant the focus is to identify the specific PKC isoforms and/or adaptor proteins involved in the PKC-mediated inhibition and to determine the relative contribution to the ACh-induced current inhibition by PKC versus the decrease in direct channel-PIP2 interactions that result due to the PIP2 hydrolysis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a revised application for partial support of the FASEB Summer Research Conference on Ion Channel Regulation (June 4-9, 2005, Snowmass Village, Colorado). Major focus of this Meeting will be on regulation of channel expression, posttranslational modification, trafficking, activity and biological function by a variety of signaling mechanisms, involving G proteins, second messengers, protein phosphorylation, cytoskeleton, membrane lipids and other. Novel signaling pathways and chanellopathies will be discussed. All the invited speakers are confirmed: they represent prominent scientists, including 6 female and 8 junior investigators, who have made important discoveries in the field of ion channel regulation. Many recent advances have occurred in ion channel regulation research, making this an important conference topic in its own right. The FASEB Conference on Ion Channel Regulation is a biennial meeting that alternates with a Gordon Conference on Ion Channels, and is designed to satisfy the growing need in significant expansion of the field and filling the gaps between channel structure and complex biological regulation, with the emphasis on signaling cascades and physiological functions. It is designed to be integrative and interdisciplinary in nature, bringing together investigators from biophysics, biochemistry, cell and molecular biology, physiology, neuroscience and medicine. This conference will highlight exciting discoveries of new families of ion channels that are regulated by novel mechanisms and serve novel biological and pathophysiological functions. More than 120 participants are expected, including pre-and postdoctoral trainees, young faculty, female and minority investigators. To promote participation of women, young investigators, and other underrepresented groups, Conference Fellowships will be offered, and they will be encouraged to present short talks that will complement each session. Requested funds will be used to provide Conference Fellowships for under-represented groups, and to partially cover travel expenses for the speakers from the U.S.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel-PIP2 interactions whereas phosphorylation by protein kinace C (PKC) leads to opposite effects. We utilized mass spectrometry to identify the phosphorylation sites within the Kir3.1 channel subunit upon treatment with protein kinases. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using MALDI-TOF/MS. Peptide peaks with a mass shift of 80u, which may relate to the addition of a phosphate group, were then subjected to tandem MS (MS2 and MS3) in order to determine the location of the modification. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to an alanyl residue resulted in a reduced sensitivity of Kir3.1* currents to H89 and forskolin, suggesting an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA. A paper describing this work has been published (Rusinova R, Shen YM, Dolios G, Padovan J, Yang H, Kirchberger M, Wang R, Logothetis DE. Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit. Pflugers Arch. 2009 Jun;458(2):303-14).

DESCRIPTION (provided by applicant): Protein phosphorylation is a common cellular mechanism used to regulate the function of most proteins. Cardiac inwardly rectifying potassium (Kir) channels are also regulated by protein phosphorylation that changes their activity and modulates cardiac excitability. Over the past ten years it has been appreciated that the activity of all Kir channels depends critically on interactions with the membrane phospholipid phosphatidylinositol-bis-phosphate (PIP2). Moreover great advances over the past five years have been made in solving the three-dimensional structures of representative Kir family members. The long term goal of our laboratory in general is to understand ion channel function and regulation in terms of molecular structure and in particular to gain mechanistic insight for the dependence of Kir activity on PIP2. We have found that many different types of Kir channel modulation, including phosphorylation, depend on channel-PIP2 interactions and we aim to understand the molecular basis of such dependence. Evidence from the literature and from our own preliminary studies suggest that phosphorylation changes the sensitivity of the channel to activation by PIP2. Examination in the three-dimensional structures of the position of putative sites that have been implicated to be involved in phosphorylation effects reveal a striking clustering around amino acid residues that affect sensitivity to PIP2. We have thus formulated the following hypothesis that we propose to test in this application: "Phosphorylation can exert its functional effects on the cardiac Kir channels by modulating channel-PIP2 interactions". Although the problem of protein phosphorylation and its mechanism of action has attracted great effort from many outstanding investigators, the experimental tools we have had to unequivocally identify single phosphorylation sites have been limiting. Thus, in the ion channel field we do not yet have mechanistic structural understanding of how phosphorylation affects channel activity. Here, we propose to use Mass Spectrometry to identify phosphorylation sites in Kir3 channels in order to test our hypothesis in a three- dimensional context. Our preliminary results have identified a protein kinase A-targeted phosphorylation site (Kir3.1-S385), using a combination of Mass Spectrometry methods (MALDI-TOF and tandem Mass Spectrometry). This result has demonstrated to us the feasibility of this approach in identifying phosphorylation sites. We propose to test electrophysiologically whether specific phosphorylation sites affect sensitivity to PIP2. A comprehensive account of sites used by different protein kinases, the assessment of which sites exert their effects through PIP2, and development of experimentally testable computational models ought to give us good mechanistic insights as to how phosphorylation regulates channel activity. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 1 Continuation Format Page. PUBLIC HEALTH RELEVANCE: Phosphorylation processes regulate cardiac performance, such as heart rate and strength of contraction, under many conditions, including exercise. This project aims to identify amino acid residues of cardiac potassium channel proteins that are phosphorylated. The hypothesis to be tested in the three-dimensional context of the proteins is that phosphorylated residues can exert their functional effects by altering directly or allosterically interactions of these channels with the key membrane phospholipid PIP2. If true, this hypothesis will provide a framework on which phosphorylation effects on channel activity could be explained mechanistically.

? DESCRIPTION (provided by applicant): 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.