Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were in'ected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of a-, A8-, r-, and 8subunit mRNAs produced two predominant channel classes with conductances of =50 and -412 pS, while infrequent openings of -25-pS channels were also observed. Injection of a-, (3-, and y-subunit mRNAs produced a single class of ==12-pS AcChoR channels, which resembled the smallest conductance channels present in apy& inected oocytes. Assembly of -less channels may thus explain the lowest conductance AcChoR channels in aPyv-i iected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle.The nicotinic acetylcholine receptor (AcChoR) of vertebrate skeletal muscle is a channel-forming pentamer thought to be composed of either a2,fy8 or aJ8e subunits. The Xenopus oocyte expression system (1-3) has been used in recent years to explore the roles of different subunits in determining receptor properties. By this means it has been shown that two principal conductance classes (40 and 60 pS) of mammalian AcChoRs differ in subunit composition, the larger conductance channel having an E subunit in place of y (4). It has also been reported that functional channels can be assembled when one of the subunits is missing. In the case of Torpedo AcChoRs, weak responses to AcCho were observed in some oocytes that were lacking y or 8 subunits (5, 6). The single channel properties of receptors lacking y or 8 subunits have not been described. Although both kinds of receptors assemble less efficiently and the 8-less receptors have reduced agonist affinity (5), the weak responses to AcCho might also reflect a reduced conductance or a briefer open time. We were interested in examining the properties ofthese receptors because they might offer an explanation for the small conductance channels (10-25 pS) observed in developing amphibian muscle (7,8). We injected mRNAs encoding the mouse a, 3, y, and 8 subunits into Xenopus oocytes and found that multiple conductance classes of channels were expressed on the oocyte membrane, and one of these, the smallest, is due to deletion of the 8 subunit. MATERIALS AND METHODSmRNA Preparation and Inection. The cDNA clones encoding mouse AcChoR subunits were generously provided by Jim Boulter (Salk Institute). mRNAs for a, (3, y, and 8 subunits were individually transcribed with SP6 polymerase. The plasmids were linearized with HindIII (clones BMA 407, BMB 49, and BMD 451 encoding a, A, and 8 subunits, respectively) or EcoRI (clone BMG 419, encoding fy subunit).The transcription reactions were carried out under standard conditions (Promega) and typically contained 2-3 ug of linearized cDNA template. The transcripts were resuspended in nuclease-free water at a concentration of 200 ng/pAl (asubunit transcript) or 100 ng/,Al (J3-, y-, and 8-subunit tran-

Epididymal adipose tissue composition and adipocyte water content were studied in male rats during growth and development of spontaneous obesity. The data show that a highly significant positive correlation exists between fat-cell volume and intracellular water space (IWS) (r=.967, P less than .001). Intracellular water, expressed as picoliters per fat cell, varied from 1.5-2 in small fat cells (mean vol, 30-50 pl) to 9-10 in large cells (800-1,000 pl). When expressed as percent of fat-cell volume, IWS varied from 5-7% in the small fat cells to 1-1.3% in the large ones. Total adipose tissue water continued to increase with increasing adipose mass. Similarly, total adipocyte water increased with enlarging cell size and tissue mass. The contribution of total adipocyte water (as contrasted to that of nonadipocyte water) to total tissue water, however, was found to be limited (less than 23%) and to decline progressively with adipose mass expansion.

The development of acetylcholine receptor (AChR) channel function in Xenopusmyotomal muscle was studied by singlechannel recordings from cell-attached patches of nonjunctional membrane in viva. AChR channels were studied from the time of their first appearance on the muscle membrane until the time of full maturity of the muscle. Two predominant amplitude classes of AChR channels were observed with slope conductances of about 40 and 60 pS. During the first day after their initial appearance on the membrane, the smallconductance channels were the most numerous class on the muscle membrane.The large-conductance channels then began to be expressed in significant numbers and, over the next 2 d, became the predominant channel type. The largeconductance class had an apparent mean open time of -0.7 msec at resting potential, which remained constant throughout development.The small channel initially had an apparent open time of -3 msec at resting potential, which decreased during development by about 50%. The decrease in open time of the small channel was correlated in time with the increased expression of the large-conductance channels. Openings of the large-conductance channels were generally separated by closed intervals of more than 1 msec, whereas openings of the small-conductance channels were commonly interrupted by brief gaps of about 0.2 msec duration. The duration of the brief gaps did not change during development.Studies of amphibian and mammalian skeletal muscle have shown that nicotinic acetylcholine receptor (AChR) channels undergo functional changes during development (reviewed by Schuetze and Role, 1987). In general, there is an increase in AChR channel conductance and a decrease in open time as the muscle matures. The first evidence of such changes came from the analysis of ACh-induced noise recorded from muscle. Recordings from muscle membrane in rats (Sakmann and Brenner, 1978;Fischbach and Schuetze, 1980) and amphibia (Kullberg et al., 198 1;Kullberg and Kasprzak, 1985) demonstrated a 3-to 4-fold decrease in the open time of AChR channels. These experiments revealed the chronology of change in gating during muscle development, but they gave no information about the conductances of the channels. With the advent of the single-

To enhance cytoplasmic delivery of liposomal contents to breast cancer cells, the authors have attached the pore-forming protein, listeriolysin O (LLO), to thermosensitive liposomes. The antibody trastuzumab (Herceptin) was also conjugated with the outer surface of the liposomes, resulting in highly specific binding and internalization into mammary epithelial cells that overexpress the human epidermal growth factor receptor 2 (Her-2). The liposomes were preloaded with a marker fluorescent dye, and the effect of LLO on the distribution of dye within the cells was monitored using fluorescence microscopy. Owing to the thermosensitive nature of the liposomes, hyperthermia at 42 degrees C triggered the release of the encapsulated fluorescent calcein from the endocytosed liposomes into the interior of the endosomes. LLO, when conjugated to these liposomes, subsequently formed pores in the endosomal membrane, allowing calcein to flow out of the endosomal compartment into the cytoplasm. Her-2-targeted liposomes bearing LLO delivered a 22-fold greater concentration of calcein to mammary epithelial cells that overexpress Her-2 compared to cells with normal Her-2 expression. Thus, the addition of LLO to preformed liposomes offers a method for significantly enhancing delivery of liposomal contents to the cytoplasm of targeted cells.

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