Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells.

The cell surfaceome is of vital importance across physiology, developmental biology, and disease states alike. The precise identification of proteins and their regulatory mechanisms at the cell membrane has been challenging and is typically determined using confocal microscopy, two-photon microscopy, or total internal reflection fluorescence microscopy (TIRFM). Of these, TIRFM is the most precise, as it harnesses the generation of a spatially delimited evanescent wave at the interface of two surfaces with distinct refractive indices.

Optogenetic dephosphorylation of phosphatidylinositol 4,5 bisphosphate in Xenopus laevis oocytes.

Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P dephosphorylation. We then detail blue light illumination to induce PI(4,5)P dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses.

Hypoxia inhibits the cardiac current through SUMO targeting Kir2.1 activation by PIP.

Cardiovascular diseases remain the leading cause of death worldwide. Most deaths are sudden and occur secondary to the occlusion of coronary arteries resulting in a rapid decrease in cellular oxygen levels. Acute hypoxia is proarrhythmic, leading to disordered electrical signals, conduction block, and uncoordinated beating of the myocardium.

A novel small-molecule selective activator of homomeric GIRK4 channels.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.

An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins.

The PKC family consists of several closely related kinases. These enzymes regulate the function of proteins through the phosphorylation of hydroxyl groups on serines and/or threonines. The selective activation of individual PKC isozymes has proven challenging because of a lack of specific activator molecules.

The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.

G-protein-gated inwardly-rectifying K (GIRK) channels are targets of G-protein-signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential.

Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP).

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP interactions.

A Critical Gating Switch at a Modulatory Site in Neuronal Kir3 Channels.

Unlabelled: Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABAB pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism.

Unifying Mechanism of Controlling Kir3 Channel Activity by G Proteins and Phosphoinositides.

The question that started with the pioneering work of Otto Loewi in the 1920s, to identify how stimulation of the vagus nerve decreased heart rate, is approaching its 100th year anniversary. In the meantime, we have learned that the neurotransmitter acetylcholine acting through muscarinic M2 receptors activates cardiac potassium (Kir3) channels via the βγ subunits of G proteins, an important effect that contributes to slowing atrial pacemaker activity. Concurrent stimulation of M1 or M3 receptors hydrolyzes PIP2, a signaling phospholipid essential to maintaining Kir3 channel activity, thus causing desensitization of channel activity and protecting the heart from overinhibition of pacemaker activity.

Gα13/PDZ-RhoGEF/RhoA signaling is essential for gastrin-releasing peptide receptor-mediated colon cancer cell migration.

Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood.

A computational model predicts that Gβγ acts at a cleft between channel subunits to activate GIRK1 channels.

The atrial G protein (heterotrimeric guanine nucleotide-binding protein)-regulated inwardly rectifying K(+) (GIRK1 and GIRK4) heterotetrameric channels underlie the acetylcholine-induced K(+) current responsible for vagal inhibition of heart rate and are activated by the G protein βγ subunits (Gβγ). We used a multistage protein-protein docking approach with data from published structures of GIRK1 and Gβγ to generate an experimentally testable interaction model of Gβγ docked onto the cytosolic domains of the GIRK1 homotetramer. The model suggested a mechanism by which Gβγ promotes the open state of a specific cytosolic gate in the channel, the G loop gate.

Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex.

Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.

Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain.

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive.

Interaction of Galphaq and Kir3, G protein-coupled inwardly rectifying potassium channels.

Activation of substance P receptors, which are coupled to Galpha(q), inhibits the Kir3.1/3.2 channels, resulting in neuronal excitation.

Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.

Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel.

Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP(2)). SP rapidly - with a half-time of approximately 10 s with intracellular GTPgammaS and approximately 14 s with intracellular GTP - inhibits a robustly activated Kir3.

Single-cell RT-PCR analysis of GIRK channels expressed in rat locus coeruleus and nucleus basalis neurons.

G protein-coupled inward rectifier potassium channels (GIRK, Kir3) play a crucial role in determining neuronal excitability. Currently, four mammalian GIRK members (GIRK1-4) have been genetically identified. We have been investigating physiological properties of GIRKs in cultured noradrenergic neurons from the locus coeruleus (LC) and cholinergic neurons from the nucleus basalis (NB).

Interaction of G protein beta subunit with inward rectifier K(+) channel Kir3.

G protein betagamma subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of Gbeta shows a seven-bladed propeller with four beta strands in each blade.

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome.

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family.