We report here the ®rst three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R). From cryo-electron microscopic images of puri®ed receptors embedded in vitreous ice, a threedimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A Ê resolution structure of the IP 3 R takes the shape of an uneven dumbbell, and is 170 A Ê tall. Its larger end is bulky, with four arms protruding laterally by~50 A Ê and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is 155 A Ê . The smaller end, whose lateral dimension is 100 A Ê , has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium¯ow in the open state of the channel.

The single-particle reconstruction problem of electron cryo-microscopy (cryo-EM) is to find the three-dimensional structure of a macromolecule given its two-dimensional noisy projection images at unknown random directions. Ab initio estimates of the 3D structure are often obtained by the "Angular Reconstitution" method, in which a coordinate system is established from three projections, and the orientation of the particle giving rise to each image is deduced from common lines among the images. However, a reliable detection of common lines is difficult due to the low signal-to-noise ratio of the images. In this paper we describe a global self-correcting voting procedure in which all projection images participate to decide the identity of the consistent common lines. The algorithm determines which common line pairs were detected correctly and which are spurious. We show that the voting procedure succeeds at relatively low detection rates and that its performance improves as the number of projection images increases. We demonstrate the algorithm for both simulative and experimental images of the 50S ribosomal subunit.

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A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.

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Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.

Several lines of evidence suggest that nonspecific drug interaction with the lipid bilayer plays an important role in subsequent recognition and binding to specific receptor sites in the membrane. The interaction of Bay K 8644, a 1,4-dihydropyridine (DHP) calcium channel agonist, with model and biological membranes was examined at the molecular level using small angle x-ray diffraction. Nonspecific drug partitioning into the membrane was examined by radiochemical assay. Nonspecific binding characteristics of [3H] Bay K 8644 were determined in both dipalmitoyl phosphatidylcholine (DPPC) vesicles above and below their thermal phase transition (Tm) and rabbit skeletal muscle light sarcoplasmic reticulum (LSR). In DPPC, the partition coefficient, Kp, was 14,000 above the Tm (55 degrees C) versus 160 in the gel phase (2 degrees C). The Kp determined in LSR membranes was 10,700. These values for both DPPC and LSR membranes can be compared with Kp = 290 in the traditional octanol/buffer system. Using small-angle x-ray diffraction, the equilibrium position of the electron-dense trifluoromethyl group of Bay K 8644 in DPPC (above Tm) and purified cardiac sarcolemmal (CSL) lipid bilayers was determined to be consistently located within the region of the first few methylene segments of the fatty acyl chains of these membranes. This position is similar to that observed for the DHP calcium channel antagonists nimodipine and Bay P 8857. We suggest this particular membrane location defines a region of local drug concentration and plane for lateral diffusion to a common receptor site. Below the DPPC membrane Tm, Bay K 8644 was shown to be excluded from this energetically favored position into the interbilayer water space. Heating the DPPC bilayer above the Tm (55 degrees C) showed that this exclusion was reversible and indicates that drug-membrane interaction is dependent on the bilayer physical state. The absence of any specific protein binding sites in these systems allows us to ascertain the potentially important role that the bulk lipid phase may play in the molecular mechanism of DHP binding to the specific receptor site associated with the calcium channel.

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