Solubilization and display of G protein-coupled receptors on beads for real-time fluorescence and flow cytometric analysis.

G protein-coupled receptors (GPCR) and cellular signaling elements are prime targets for drug discovery. Sensitive real-time methods that expand the analytical capabilities for these elements can play significant roles in basic research and drug discovery. Here, we describe novel approaches for the real-time fluorescence analysis of GPCRs. Using the G protein-coupled N-formyl peptide receptor (FPR) as a model system in concert with a fluorescent ligand, we showed the quantitative solubilization of his-tagged FPRs in 1% dodecyl maltoside. Solubilized receptors reconstitute in dodecyl maltoside with a mixture of bovine brain Gi/Go showing an apparent Kd of 100 nM. Solubilized receptors were also bound to Ni(2+)-silica particles and were detected in a flow cytometer by the binding of fluorescent ligand. The efficiency of receptor uptake by the particles was in excess of 80% with an apparent affinity for the bead in the nM range. The receptors had largely homogeneous dissociation characteristics, an appropriate Kd for the ligand in the low nM range and a high site number, with several million receptor molecules per particle. However, the G protein reconstitution was not detected on the beads, apparently for steric reasons. These approaches for displaying receptors could prove useful in drug discovery and in the analysis of the molecular assemblies in signal transduction.

Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands.

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.

Abnormal ryanodine receptor channels in malignant hyperthermia.

Previous studies have demonstrated a defect associated with the calcium release mechanism of sarcoplasmic reticulum (SR) from individuals susceptible to malignant hyperthermia (MH). To examine whether SR calcium release channels were indeed altered in MH, SR vesicles were purified from normal and MH susceptible (MHS) porcine muscle. The Ca2+ dependence of calcium efflux rates from 45Ca2(+)-filled SR vesicles was then compared with the Ca2+ dependence of single-channel recordings of SR vesicles incorporated into planar lipid bilayers. The rate constants of 45Ca2+ efflux from MHS SR were two to threefold larger than from normal SR over a wide range of myoplasmic Ca2+. Normal and MHS single channels were progressively activated in a similar fashion by cis Ca2+ from pCa 7 to 4. However, below pCa 4, normal channels were inactivated by cis Ca2+, whereas MHS channels remained open for significantly longer times. The altered Ca2+ dependence of channel inactivation in MHS SR was also evident when Ca2+ was increased on the trans side while cis Ca2+ was held constant. We propose that a defect in a low-affinity Ca2+ binding site is responsible for the altered gating of MHS SR channels. Such a defect could logically result from a mutation in the gene encoding the calcium release channel, providing a testable hypothesis for the molecular basis of this inherited disorder.

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate.

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.

Calcium channel activity in a purified dihydropyridine-receptor preparation of skeletal muscle.

A purified dihydropyridine-receptor complex (DHPR) of skeletal muscle consisting of a major polypeptide of Mr 150K under reducing conditions induces divalent cation selective channels when incorporated into planar lipid bilayers. Channels were inserted into preformed planar bilayers by two techniques: (i) direct dilution of detergent-solubilized DHPR into the aqueous chambers adjacent to the bilayer membrane or (ii) reconstitution of DHPR into phospholipid vesicles followed by fusion of the preformed vesicles to the planar bilayer membrane. Unlike native membrane preparations of t-tubules, which only have one major Ca channel type of slope conductance of 12 pS in symmetrical 100 mM Ba, the purified DHPR complex induced at least two channel types with conductances of 12-14 and 22 pS. Some recordings suggest that these two channels are statistically coupled in time, i.e., that they may correspond to substrates of the same DHPR channel. Activity was found to occur spontaneously in the absence of the Ca channel agonist Bay k 8644. The 12-14-pS channel from DHPR exhibits voltage-dependent kinetics, is highly selective for barium ions, and was inhibited by micromolar nitrendipine. The 12-14-pS DHPR channel appears to be identical with functional Ca channels previously described in native t-tubules.