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Putting G protein-coupled receptor-mediated activation of phospholipase C in the limelight

Balla, T. — Mon, 25 Jan 2010 09:06:57 PST

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

Falkenburger, B. H., Jensen, J. B., Hille, B. — Mon, 25 Jan 2010 09:06:57 PST

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the G subunit, are common for many different GPCRs. For G_q-coupled M₁ muscarinic (acetylcholine) receptors (M₁Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M₁R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M₁R signaling through G_q to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and PIP₂-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ~3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm² of plasma membrane. Endogenous levels are much lower, 1–40 per µm². Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M₁R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to G_q greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on G_q subunits bound to PLC.

Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

Falkenburger, B. H., Jensen, J. B., Hille, B. — Mon, 25 Jan 2010 09:06:57 PST

The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP₂) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP₂ pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP₂ with a time constant of <300 ms and fully inhibits KCNQ currents in a similar time. PIP₂ was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, = 11 s. In contrast, resynthesis of PIP₂ after activation of phospholipase C by muscarinic receptors took ~130 s. These kinetic experiments showed that (1) PIP₂ activation of KCNQ channels obeys a cooperative square law, (2) the PIP₂ residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP₂ by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M₁ muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP₂ synthesis and KCNQ interaction.

Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit

Meier, S., Tavraz, N. N., Durr, K. L., Friedrich, T. — Mon, 25 Jan 2010 09:06:57 PST

The Na⁺/K⁺-ATPase mediates electrogenic transport by exporting three Na⁺ ions in exchange for two K⁺ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb⁺ ions in the crystal structures of the Na⁺/K⁺-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na⁺ or K⁺ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na⁺ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na⁺/K⁺-ATPase ₂ subunit decreases the affinity for extracellular and intracellular Na⁺, in agreement with previous biochemical studies. Apparently, the YY deletion changes Na⁺ affinity at site III but leaves the common sites unaffected, whereas the more extensive KETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K⁺, the YY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na⁺ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na⁺/Na⁺ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na⁺ release/binding mechanism. In analogy to the mechanism proposed for the H⁺ leak currents of the wild-type Na⁺/K⁺-ATPase, we suggest that the YY deletion lowers the energy barrier for the intracellular Na⁺ occlusion reaction, thus destabilizing the Na⁺-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na⁺/K⁺-ATPase subunit represents a structural and functional relay between Na⁺ binding site III and the intracellular cation occlusion gate.

Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences

Mon, 25 Jan 2010 09:06:57 PST

Voltage-dependent potassium (K_v) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K_v channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K⁺ currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K_v1.3/K_v1.5 channels, which generate the main K_v in macrophages. An increase in K⁺ current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K_v1.3 subunits in the K_v1.3/K_v1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K_v1.3 expression. Neither of these treatments apparently altered the expression of K_v1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K_v1.3/K_v1.5 hybrid channels by altering the subunit stoichiometry.

Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore

Martinez-Francois, J. R., Lu, Z. — Mon, 25 Jan 2010 09:06:57 PST

Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide–gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller "diameter" and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation

Findeisen, F., Minor, D. L. — Mon, 25 Jan 2010 09:06:57 PST

Comparison of regulated passive membrane conductance in action potential-firing fast- and slow-twitch muscle

Mon, 25 Jan 2010 09:06:57 PST