William E. McIntire

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

Differential Sensitivity of P-Rex1 to Isoforms of G Protein βγ Dimers

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gβγ subunits of heterotrimeric G proteins. We examined which of the multiple G protein α and βγ subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF–4 activated Gs,Gi,Gq,G12, or G13 α subunits were unable to activate P-Rex1. Gβγ dimers containing Gβ1–4 complexed with γ2 stimulated P-Rex1 activity with EC50 values ranging from 10 to 20 nm. Gβ5γ2 was not able to stimulate P-Rex1 GEF activity. Dimers containing the β1 subunit complexed with a panel of different Gγ subunits varied in their ability to stimulate P-Rex1. The β1γ3, β1γ7, β1γ10, and β1γ13HA dimers all activated P-Rex1 with EC50 values ranging from 20 to 38 nm. Dimers composed of β1γ12 had lower EC50 values (∼112 nm). The farnesylated γ11 subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (β1γ11) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gβγ dimer released by receptor activation may differentially activate P-Rex1.

The molecular basis for T-type Ca2+ channel inhibition by G protein β2γ2 subunits

Gβγ, a ubiquitous second messenger, relays external signals from G protein-coupled receptors to networks of intracellular effectors, including voltage-dependent calcium channels. Unlike high-voltage-activated Ca2+ channels, the inhibition of low-voltage-activated Ca2+ channels is subtype-dependent and mediated selectively by Gβ2-containing dimers. Yet, the molecular basis for this exquisite selectivity remains unknown. Here, we used pure recombinant Gβγ subunits to establish that the Gβ2γ2 dimer can selectively reconstitute the inhibition of α1H channels in isolated membrane patches. This inhibition is the result of a reduction in channel open probability that is not accompanied by a change in channel expression or an alteration in active-channel gating. By exchanging residues between the active Gβ2 subunit and the inactive Gβ1 subunit, we identified a cluster of amino acids that functionally distinguish Gβ2 from other Gβ subunits. These amino acids on the β-torus identify a region that is distinct from those regions that contact the Gα subunit or other effectors.

Protein kinase A activity controls the regulation of T-type CAV3.2 channels by Gβγ dimers

Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gβγ dimers, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gβ2γx dimers to effect voltage-independent inhibition of Cav3.2 channels. Inhibition requires phosphorylation of Ser1107, a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gβ2γ2 dimers but does not disrupt dimer binding nor change its specificity. Gβγ dimers released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Cav3.2 whole cell current is precluded by Gβγ-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, D1 or D2 agonists do not elicit channel inhibition yet together synergize to inhibit Cav3.2 channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Cav3.2 channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.

Inhibition of Mammalian Gq Protein Function by Local Anesthetics

Background Local anesthetics have been shown to selectively inhibit functioning of Xenopus laevis Gq proteins. It is not known whether a similar interaction exists with mammalian G proteins. The goal of this study was to determine whether mammalian Gq protein is inhibited by local anesthetics. Methods In Xenopus oocytes, the authors replaced endogenous Gq protein with mouse Gq (expressed in Sf9 cells using baculovirus vectors). Cells endogenously expressing lysophosphatidic acid or recombinantly expressing muscarinic m3 receptors were injected with phosphorothioate DNA antisense (or sense as control) oligonucleotides against Xenopus Gq. Forty-eight hours later, oocytes were injected with purified mouse Gq (5 × 10−8 m) or solvent as control. Two hours later, the authors injected either lidocaine, its permanently charged analog QX314 (at IC50, 50 nl), or solvent (KCl 150 mm) as control and measured Ca-activated Cl currents in response to lysophosphatidic acid or methylcholine (one tenth of EC50). Results Injection of anti-Gq reduced the mean response size elicited by lysophosphatidic acid to 33 ± 7% of the corresponding control response. In contrast, responses were unchanged (131 ± 29% of control) in cells in addition injected with mouse Gq protein. Injection of mouse Gq protein “rescued” the inhibitory effect of intracellularly injected QX314: whereas QX314 was without effect on Gq-depleted oocytes, responses to lysophosphatidic acid after QX314 injection were inhibited to 44 ± 10% of control response in cells in addition injected with mouse Gq protein (5 × 10−8 m). Similar results were obtained for m3 signaling and intracellularly injected lidocaine. Conclusion Inhibition of Gq function by local anesthetics is not restricted to Xenopus G proteins. Therefore, Gq should be considered as one additional intracellular target site for local anesthetics, especially relevant for those effects not explainable by sodium channel blockade (e.g., antiinflammatory effects).

Reconstitution of G protein-coupled receptors with recombinant G protein α and βγ subunits

Publisher Summary This chapter describes experiments that can probe the first steps in receptor–G protein interaction using defined, recombinant receptors and G proteins. A basic issue in cell signaling is the identification of those receptors that interact with specific isoforms of the multiple G protein α and βγ subunits. At the molecular level, it is important to understand which domains of the G protein α and βγ subunits interact with the intracellular loops and transmembrane helices of receptors. Thus, it is useful to have methods to study the receptor–G protein interaction with recombinant proteins. To date, many techniques have been developed to examine this question, which use intact cells, isolated plasma membranes, and/or purified receptors, and G proteins. The protocols have the advantages that the receptors are inserted properly in a cell membrane and that the investigator has complete control of the proteins reconstituted with the receptor. Specific mutations in the receptors or G proteins are studied easily and the protocols allow precise examination of the stoichiometry of the receptor–α–βγ interaction.

Function and dynamics of macromolecular complexes explored by integrative structural and computational biology

Three vignettes exemplify the potential of combining EM and X-ray crystallographic data with molecular dynamics (MD) simulation to explore the architecture, dynamics and functional properties of multicomponent, macromolecular complexes. The first two describe how EM and X-ray crystallography were used to solve structures of the ribosome and the Arp2/3-actin complex, which enabled MD simulations that elucidated functional dynamics. The third describes how EM, X-ray crystallography, and microsecond MD simulations of a GPCR:G protein complex were used to explore transmembrane signaling by the β-adrenergic receptor. Recent technical advancements in EM, X-ray crystallography and computational simulation create unprecedented synergies for integrative structural biology to reveal new insights into heretofore intractable biological systems.

Functional TASK-3–Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells

Ca2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K+ channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K+ channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells.