Ca2+ channel-independent requirement for MAGUK family CACNB4 genes in initiation of zebrafish epiboly.

CACNB genes encode membrane-associated guanylate kinase (MAGUK) proteins once thought to function exclusively as auxiliary beta subunits in assembly and gating of voltage-gated Ca(2+) channels. Here, we report that zygotic deficiency of zebrafish beta4 protein blocks initiation of epiboly, the first morphogenetic movement of teleost embryos. Reduced beta4 function in the yolk syncytial layer (YSL) leads to abnormal division and dispersal of yolk syncytial nuclei, blastoderm retraction, and death, effects highly similar to microtubule disruption by nocodazole. Epiboly is restored by coinjection of human beta4 cRNA or, surprisingly, by mutant cRNA encoding beta4 subunits incapable of binding to Ca(2+) channel alpha1 subunits. This study defines a YSL-driven zygotic mechanism essential for epiboly initiation and reveals a Ca(2+) channel-independent beta4 protein function potentially involving the cytoskeleton.

Primate anesthesia.

This article provides an update on some of the recent advances in primate anesthesia. It focuses in particular on some of the newest information available regarding the effects of opioids and alpha-2 agonists in primates, and how these effects are different from what we might expect in other companion animals. It reviews the important properties of the latest induction and inhalation agents, and stresses the need for continuous monitoring of the anesthetized patient.

A simple method of providing intermittent positive-pressure ventilation to etorphine-immobilized elephants (Loxodonta africana) in the field.

Five African elephants (Loxodonta africana) were immobilized with etorphine in Waza National Park, Cameroon, for the purpose of deploying radio/satellite tracking collars. A portable ventilator constructed from two high-flow demand valves and the Y-piece of a large animal anesthesia circuit was used to provide intermittent positive-pressure ventilation with 100% oxygen. Oxygenation status improved dramatically in all five elephants. In one hypoxemic elephant, arterial PaO2 increased from 40 to 366 mm Hg. The results of this study demonstrate that both oxygenation and ventilation can be readily controlled in etorphine-immobilized elephants even under remote field conditions.

Capnographic monitoring of anesthetized African grey parrots receiving intermittent positive pressure ventilation.

OBJECTIVE: To determine whether end-tidal partial pressure of carbon dioxide (PETCO2) correlated with PaCO2 in isoflurane-anesthetized African grey parrots receiving intermittent positive pressure ventilation (IPPV). DESIGN: Prospective study. ANIMALS: 14 healthy mature African grey parrots (Psittacus erithacus timnus). PROCEDURE: Each bird was anesthetized via mask with isoflurane, intubated, and connected to a pressure-limited intermittent-flow ventilator. Respiratory rate was altered while holding peak inspiratory pressure constant (5 cm H2O) to achieve a PETCO2 in 1 of 3 ranges: < 30 mm Hg, 30 to 40 mm Hg, and > 40 mm Hg. Blood was collected from the superficial ulnar artery of each bird at least once during each of the 3 ranges. Arterial blood samples were collected for blood gas analysis while PETCO2 was recorded simultaneously. RESULTS: A strong correlation between PETCO2 and PaCO2 was detected over a wide range of partial pressures, although PETCO2 consistently overestimated PaCO2 by approximately 5 mm Hg. End-tidal partial pressure of CO2 and PaCO2 also correlated well with arterial blood pH, and the acute response of the bicarbonate buffer system to changes in ventilation was similar to that of mammals. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that PETCO2 reliably estimates PaCO2 in isoflurane-anesthetized African grey parrots receiving IPPV and suggest that IPPV combined with capnography is a viable option for anesthetic maintenance in avian anesthesia.

Cardiorespiratory effects of four alpha2-adrenoceptor agonist-ketamine combinations in captive red wolves.

OBJECTIVE: To evaluate the cardiopulmonary effects of immobilizing doses of xylazine-ketamine (XK), medetomidine-ketamine (MK), medetomidine-ketamine-acepromazine (MKA), and medetomidine-butorphanol-ketamine (MBK) in captive red wolves. DESIGN: Prospective study. ANIMALS: 32 adult captive red wolves. PROCEDURE: Wolves were randomly assigned to 1 of 4 treatment groups: XK, MK, MKA, or MBK. Physiologic variables measured included heart rate, blood pressure, respiratory rate, tidal volume, oxygen-hemoglobin saturation (Spo2), end-tidal CO2, arterial blood gases, and rectal temperature. Induction time, muscle relaxation, and quality of recovery were assessed. RESULTS: Heart rates were lower in wolves in the MBK group than for the other groups. All 4 drug combinations induced considerable hypertension, with diastolic pressures exceeding 116 mm Hg. Blood pressure was lowest in wolves receiving the MBK combination. Respiratory rate was significantly higher in wolves receiving XK, MK, and MKA. Tidal volumes were similar for all groups. Wolves receiving XK, MK, and MKA were well-oxygenated throughout the procedure (SPo2 > 93%), whereas those receiving MBK were moderately hypoxemic (87% < Spo2 < 93%) during the first 20 minutes of the procedure. Hyperthermia was detected initially following induction in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: The alpha2-adrenoceptor agonist-ketamine combinations provide rapid reversible anesthesia for red wolves but cause severe sustained hypertension. Such an adverse effect puts animals at risk for development of cerebral encephalopathy, retinal hemorrhage, pulmonary edema, and myocardial failure. Although the MBK combination offers some advantages over the others, it is advised that further protocol refinements be made to minimize risks associated with acute hypertension.

C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit.

The calcium channel alpha(1A) subunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The alpha(1A) C terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of alpha(1A) subunits. Specifically, the biophysical properties of two alternatively spliced alpha(1A) subunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression in Xenopus oocytes demonstrated that the two variants were similar with respect to current-voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which alpha(1A) subunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca(2+) channel function.

Cloning and sequencing hybrid striped bass (Morone saxatilis x M. chrysops) transforming growth factor-beta (TGF-beta), and development of a reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay to measure TGF-beta mRNA of teleost fish.

A transforming growth factor (TGF)-beta was isolated and cloned from hybrid striped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclear cells. This isolate (Genbank accession number AF140363) contains an open reading frame of 1146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF-beta (57.3 and 78.6% identity with precursor and active protein, respectively) and rat TGF-beta 1 (41.1 and 68.8% identity with precursor and active protein, respectively). Consensus primers were demonstrated to amplify specifically by polymerase chain reaction (PCR), a TGF-beta segment from 14 species of teleost fish comprising 10 taxonomic families in 7 orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-beta mRNA expression in teleost fish. Higher levels of TGF-beta mRNA expression were detected in mononuclear cells of peripheral blood than from spleen or anterior kidney.

Evaluation of epidural morphine for postoperative analgesia in ferrets (Mustela putorius furo).

We evaluated the analgesic efficacy of epidural morphine for relieving postoperative pain in domestic ferrets by evaluating behavior and fecal cortisol concentrations. The 12 laboratory-reared, intact, female, domestic ferrets were anesthetized then underwent ovariohysterectomy and bilateral anal sacculectomy. Using a double-blind procedure, we provided epidural morphine (0.1 mg/kg) to six ferrets and epidural saline (0.1 mL/ferret) to the remaining animals prior to surgery. Compared to the animals that received saline, the morphine-treated ferrets were more likely to have attenuated pain responses, and they returned more rapidly to preoperative behavior. Although fecal cortisol concentrations during the first 24 h after surgery increased in all animals, the increase was statistically significant only in the ferrets that received saline epidurals. These data suggest that morphine epidurals administered to ferrets prior to surgery may attenuate both the physiologic and behavioral manifestations of surgically induced pain.

Overexpression of the Bcl-2 protein increases the half-life of p21Bax.

Bcl-2 and Bax are homologous proteins which can heterodimerize with each other. These proteins have opposing effects on cell survival when overexpressed in cells, with Bcl-2 blocking and Bax promoting apoptosis. Here we demonstrate that gene transfer-mediated elevations in Bcl-2 protein levels result in a marked increase in the steady-state levels of endogenous p21Bax protein as determined by immunoblotting in the Jurkat T-cell and 697 pre-B-cell leukemia cell lines, but not in several other cell lines including CEM T-cell leukemia, 32D.3 myeloid progenitor, PC12 pheochromocytoma, and NIH-3T3 fibroblasts. Steady-state levels of p21Bax protein were also elevated in the lymph nodes of Bcl-2 transgenic mice in which a BCL-2 transgene is expressed at high levels in B-cells. Northern blot analysis of BCL-2-transfected and control-transfected Jurkat and 697 leukemia cells revealed no Bcl-2-induced alterations in the steady-state levels of BAX mRNAs. In contrast, L-[35S]methionine pulse-chase analysis indicated a marked increase in the half-life (t1/2) of the p21Bax protein in BCL-2-transfected 697 cells compared to control-transfected cells (t1/2 > 24 h versus approximately 4 h), whereas the rate of Bax degradation was unaltered in Bcl-2-transfected CEM cells. The results demonstrate that levels of the proapoptotic p21Bax protein can be post-translationally regulated by Bcl-2, probably in a tissue-specific fashion, and suggest the existence of a feedback mechanism that may help to maintain the ratio of Bcl-2 to Bax protein in physiologically appropriate ranges.

Structural determinants of the blockade of N-type calcium channels by a peptide neurotoxin.

Neurotoxins that selectively block Na+, K+ or Ca2+ channels have provided valuable information about the functional diversity of the voltage-gated channel superfamily. For Ca2+ channels, a variety of toxins have been found to block individual channel types. The best-known example is omega-conotoxin-GVIA, a member of a large family of peptide toxins derived from venomous cone snails, which potently and selectively blocks N-type Ca2+ channels, allowing their purification, cellular localization, and the elucidation of their roles in Ca2+ entry, neurotransmitter release and neuronal migration. In contrast to Na+ and K+ channels, little is known about the molecular features that underlie Ca(2+)-channel susceptibility to toxin block; it is also unknown whether block occurs by direct physical occlusion or an action on channel gating. Here we describe structural determinants of N-type Ca2+ channel's interaction with omega-conotoxin-GVIA. When chimaeras combining individual motifs from the N-type channel and from a channel insensitive to omega-conotoxin-GVIA were expressed in Xenopus oocytes, each of the four motifs appeared to contribute to interaction with the toxin. The most dramatic effects on toxin interactions were seen at a single cluster of residues in the large putative extracellular loop between IIIS5 and IIIH5, consistent with a direct pore-blocking mechanism. These results provide a starting point for delineating the architecture of the outer vestibule of the Ca2+ channel.

Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons.

This paper provides a brief overview of the diversity of voltage-gated Ca2+ channels and our recent work on neuronal Ca2+ channels with novel pharmacological and biophysical properties that distinguish them from L, N, P or T-type channels. The Ca2+ channel alpha 1 subunit known as alpha 1A or BI [Mori Y., Friedrich T., Kim M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T. and Numa S. (1991) Nature 350, 398-402] is generally assumed to encode the P-type Ca2+ channel. However, we find that alpha 1A expressed in Xenopus oocytes differs from P-type channels in its kinetics of inactivation and its degree of sensitivity to block by the peptide toxins omega-Aga-IVA and omega-CTx-MVIIC [Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Neuron 11, 291-303]. Thus, alpha 1A is capable of generating a Ca2+ channel with characteristics quite distinct from P-type channels. Doe-1, recently cloned from the forebrain of a marine ray, is another alpha 1 subunit which exemplifies a different branch of the Ca2+ channel family tree [Horne W. A., Ellinor P. T., Inman I., Zhou M., Tsien R. W. and Schwarz T. L. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 3787-3791]. When expressed in Xenopus oocytes, doe-1 forms a high voltage-activated (HVA) Ca2+ channel [Ellinor P. T., Zhang J.-F., Randall A. D., Zhou M., Schwarz T. L., Tsien R. W. and Horne W. (1993) Nature 363, 455-458]. It inactivates more rapidly than any previously expressed calcium channel and is not blocked by dihydropyridine antagonists or omega-Aga-IVA. Doe-1 current is reduced by omega-CTx-GVIA, but the inhibition is readily reversible and requires micromolar toxin, in contrast to this toxin's potent and irreversible block of N-type channels. Doe-1 shows considerable sensitivity to block by Ni2+ or Cd2+. We have identified components of Ca2+ channel current in rat cerebellar granule neurons with kinetic and pharmacological features similar to alpha 1A and doe-1 in oocytes [Randall A. D., Wendland B., Schweizer F., Miljanich G., Adams M. E. and Tsien R. W. (1993) Soc. Neurosci. Abstr. 19, 1478]. The doe-1-like component (R-type current) inactivates much more quickly than L, N or P-type channels, and also differs significantly in its pharmacology.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional expression of a rapidly inactivating neuronal calcium channel.

Diverse types of calcium channels in vertebrate neurons are important in linking electrical activity to transmitter release, gene expression and modulation of membrane excitability. Four classes of Ca2+ channels (T, N, L and P-type) have been distinguished on the basis of their electrophysiological and pharmacological properties. Most of the recently cloned Ca2+ channels fit within this functional classification. But one major branch of the Ca2+ channel gene family, including BII (ref. 15) and doe-1 (ref. 16), has not been functionally characterized. We report here the expression of doe-1 and show that it is a high-voltage-activated (HVA) Ca2+ channel that inactivates more rapidly than previously expressed calcium channels. Unlike L-type or P-type channels, doe-1 is not blocked by dihydropyridine antagonists or the peptide toxin omega-Aga-IVA, respectively. In contrast to a previously cloned N-type channel, doe-1 block by omega-CTx-GVIA requires micromolar toxin and is readily reversible. Unlike most HVA channels, doe-1 also shows unusual sensitivity to block by Ni2+. Thus, doe-1 is an HVA Ca2+ channel with novel functional properties. We have identified a Ca2+ channel current in rat cerebellar granule neurons that resembles doe-1 in many kinetic and pharmacological features.

Molecular diversity of Ca2+ channel alpha 1 subunits from the marine ray Discopyge ommata.

In many neurons, transmitter release from presynaptic terminals is triggered by Ca2+ entry via dihydropyridine-insensitive Ca2+ channels. We have looked for cDNAs for such channels in the nervous system of the marine ray Discopyge ommata. One cDNA (doe-2) is similar to dihydropyridine-sensitive L-type channels, and two cDNAs (doe-1 and doe-4) are similar to the subfamily of dihydropyridine-insensitive non-L-type channels. doe-4, which encodes a protein of 2326 aa, most closely resembles a previously cloned N-type channel. doe-1, which encodes a protein of 2223 aa, is a member of a separate branch of the non-L-type channels. Northern blot analysis reveals that doe-1 is abundant in the forebrain. doe-4 is more plentiful in the electric lobe and, therefore, may control neurotransmitter release in motor nerve terminals. These results show that the familial pattern of Ca(2+)-channel genes has been preserved from a stage in evolution before the divergence of higher and lower vertebrates > 400 million years ago. The cloning of these channels may be a useful starting point for elucidating the role of the Ca2+ channels in excitation-secretion coupling in nerve terminals.

Molecular diversity of voltage-dependent Ca2+ channels.

Voltage-dependent Ca2+ channels regulate Ca2+ entry and thereby contribute to Ca2+ signalling in many cells. Functional studies have uncovered several types of Ca2+ channel, distinguished by pharmacology, electrophysiology and tissue localization. More recently, molecular cloning has revealed an even greater diversity among Ca2+ channels, arising from multiple genes and alternative splicing. L-type, dihydropyridine-sensitive Ca2+ channels have been the most extensively characterized to date. Recently, Numa's group has reported the cloning and expression of a dihydropyridine-insensitive Ca2+ channel from brain that most closely resembles the P-type channel described by Llinas and colleagues. These results contribute to rapidly growing knowledge about molecular determinants of Ca2+ channel diversity.

omega-Conotoxin GVIA receptors of Discopyge electric organ. Characterization of omega-conotoxin binding to the nicotinic acetylcholine receptor.

A peptide toxin from a Conus marine snail, omega-conotoxin GVIA (omega-CgTx) has been used extensively as a probe for certain types of neuronal calcium channels. It is often assumed that omega-CgTx interacts with Ca2+ channels exclusively. We have tested this assumption in a study of omega-CgTx-binding sites in the electric organ of Discopyge ommata. Synaptosomal membranes from this tissue contain low affinity omega-CgTx receptor sites (Kd = 0.6 microM) in great abundance (280 pmol/mg of protein), as first reported by Ahmad and Miljanich (Ahmad, S. N., and Miljanich, G.P. (1988) Brain Res. 453, 247-256). However, we find that a large majority of these omega-CgTx-binding sites co-purify with the nicotinic acetylcholine receptor (nAChR) and can be immunoprecipitated by monoclonal antibodies generated against the nAChR of Torpedo. Cross-linking experiments with radiolabeled omega-CgTx show pronounced specific labeling of the alpha-subunit of the nAChR but not other subunits. Specific omega-CgTx binding to the nAChR is reduced by millimolar Ca2+ but not by alpha- or kappa-bungarotoxin, alpha-conotoxin, or carbamylcholine. Cross-linking experiments also reveal omega-CgTx-binding proteins of 170 and 60 kDa. The characteristics of the 170-kDa protein make it a likely candidate for the alpha 1-subunit of an N-type Ca2+ channel.

Functional reconstitution of skeletal muscle Ca2+ channels: separation of regulatory and channel components.

Regulatory properties of a partially purified Ca2+ -channel preparation from isolated rabbit skeletal muscle triads were examined in proteoliposomes. These properties included (i) inhibition by phenylalkylamine antagonists, such as verapamil, (ii) inhibition by the GTP-binding protein Go in the presence of guanosine 5'-[gamma-thio]triphosphate, and (iii) regulation of phenylalkylamine inhibition as a result of phosphorylation by a polypeptide-dependent protein kinase (PK-P). By selective reconstitution of protein fractions obtained by wheat germ lectin and ion-exchange chromatography, a separation of Ca2+-channel activity (fraction C) from regulatory component(s) (fraction R) responsible for verapamil sensitivity was achieved. Reconstitution of fraction C alone yielded vesicles that exhibited channel-mediated 45Ca2+ uptake that could be directly inhibited by coreconstitution of Go in the presence of guanosine 5'-[gamma-thio]triphosphate. However, the 45Ca2+ uptake obtained with fraction C was not inhibited by verapamil. Coreconstitution of fractions C and R yielded vesicles in which the sensitivity of 45Ca2+ uptake to verapamil was restored. The verapamil sensitivity of this preparation could be inhibited by PK-P. Fraction C, obtained by wheat germ agglutinin-Sepharose chromatography followed by DEAE-Sephacel chromatography, included a 180-kDa protein that was phosphorylated by cAMP-dependent protein kinase (PK-A) but not by PK-P and a 145-kDa protein (180 kDa under nonreducing conditions) that was not phosphorylated by either kinase. Fraction R contained proteins that did not adsorb to wheat germ lectin and included 165-kDa and 55-kDa proteins that were phosphorylated by PK-P but not by PK-A. These results suggest a complex model for Ca2+-channel regulation in skeletal muscle involving a number of distinct, separable protein components.

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles.

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.

Repeatability of the M-mode echocardiogram and the effects of acute changes in heart rate, cardiac contractility, and preload in healthy cats sedated with ketamine hydrochloride and acepromazine.

In healthy cats sedated with a combination of ketamine hydrochloride and acepromazine, the reproducibility of sequential echocardiographic examinations was studied. The effects of experimental alteration in heart rate, contractility, and preload on the echocardiogram also were studied. Ten cats had M-mode echocardiograms recorded once weekly for 5 weeks. There were no statistically significant differences in echocardiographic measurements over time, although measurements of certain cardiac structures of an individual cat did vary from time to time. The cardiovascular system was altered by a constant infusion of isoproterenol, followed by a rapid intravenous infusion of isotonic fluid equal to 25% of the calculated blood volume, and then by a rapid withdrawal of blood equal to the amount of fluid previously given. Cardiac output using indocyanine green dilution curves were determined for each treatment and compared to the cardiac output calculated from the echocardiogram. The correlation coefficient was 0.90 for all treatments. During the infusion of isoproterenol a significant increase in the percent fractional shortening and left ventricular wall amplitude occurred compared to the control echocardiogram. After the rapid infusion of fluid, none of the mean echocardiographic measurements significantly changed from control values although the left ventricular chamber diameter at systole significantly increased, and the percent fractional shortening significantly decreased, when compared to values obtained during isoproterenol infusion. Significant changes recorded between echocardiographic measurements made after blood withdrawal compared to those made after fluid loading included an increase in left ventricular wall thickness at diastole, a decrease in left ventricular diameter at diastole, and a decrease in the present left ventricular wall thickening. Cats were terminated and the average of measurements made during the repeatability study of the septal and left ventricular wall thickness during systole, rather than during diastole, more closely approximated the postmortem measurements.

Solubilization and hydrodynamic characterization of the dihydropyridine receptor from rat ventricular muscle.

The dihydropyridine receptor-calcium channel complex, prelabeled with (+)-[3H]PN200-110, was solubilized from rat heart membranes with a detergent mixture of digitonin and Triton X-100. The dissociation of (+)-[3H]PN200-110 was slow enough to permit the hydrodynamic characterization of the complex by means of sucrose gradient sedimentation and gel filtration. The hydrodynamic properties of the complex were determined in several detergents, including Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and digitonin. S20,w values of 12.5, 15.4, and 21.0 S were obtained in sucrose gradients prepared in Tween 80, CHAPS, and digitonin, respectively. A Stokes radius of 86-87 A was obtained in each of the three detergents. Determination of the partial specific volume of the protein-detergent complex in each case revealed that the differences in S20,w values could be explained by the differences in the properties of the bound detergent species. Partial specific volumes of 0.796, 0.730, and 0.730 ml/g, corresponding to molecular weights of 595,000, 540,000, and 740,000 were obtained for the complex in Tween 80, CHAPS, and digitonin, respectively. This indicated that Tween 80 readily exchanged for the solubilizing mixture of digitonin and Triton X-100, whereas CHAPS did not. Detergent exchange with Tween 80 made it possible to determine the fractional contribution of the receptor protein to the molecular weight of the protein-detergent complex. The molecular weight of the dihydropyridine receptor-calcium channel complex was estimated to be 370,000. The protein-detergent complex was found to have a frictional coefficient of 1.39, consistent with a large transmembrane protein.