The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site . This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton . If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2 :1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.A large variety of cellular events including mitosis, cell elongation, and neurite outgrowth are believed to be related to the polymerization and depolymerization of microtubules in vivo (1) . However, the mechanisms by which these events might be regulated are only begining to be understood, with the bulk of the evidence drawn from in vitro biochemical studies .A number ofinvestigators (2) have found that microtubules in crude extracts are depolymerized by concentrations of calcium much lower than those required to depolymerize tubules made from purified microtubule proteins. This disparity suggested that some factor in crude extracts "sensitized" the tubules to calcium ; calmodulin was an obvious candidate for this function . When calmodulin was added to purified microtubule proteins, Marcum et al. (3) found that the calcium concentration required for microtubule depolymerization was decreased by two orders of magnitude, to approximately the level observed in crude extracts. In addition it was found that the inhibition of microtubule polymerization was proportional both to calmodulin and to calcium concentrations (3) . While this interaction occurs at calmodulin:tubulin in ratios much higher than the ratios of calmodulin :enzyme necessary to activate cellular enzymes, at least two factors argue in favor of its physiological relevance. First, the im- 1918CHARLES KEITH, MARIO DiPAOLA, FREDERICK R. MAXFIELD, and MICHAEL L. SHELANSKI Department of Pharmacology, New York University Medical Center, New York 10016 munocytochemical localization of calmodulin between the chromosomes and spindle poles of mitotic cells (4, 5) and at the postsynaptic termini of basal ganglion neurons (6) correlates with the locations in the cell where microtubule depolymerization might be expected to occur...

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The nicotinic acetylcholine receptor is a multisubunit, membrane-spanning protein that contains a gated, cation-conducting channel. Our approach to the understanding of the function of this receptor in molecular terms has been to locate its functionally significant sites in the sequences of its subunits and in its three-dimensional structure. In addition, we have tried to correlate transitions in the properties of these sites with functional transitions of the receptor. On binding acetylcholine, the nicotinic acetylcholine receptor enters at least two transient states, the open state and the rapid-onset desensitized state, and, in the continued presence of agonist, finally subsides into the slow-onset desensitized state. The transitions of the receptor between these various states are susceptible to regulation by acetylcholine and its congeners acting at one type of site and by a broad class of noncompetitive inhibitors (NCIs), including local anesthetics, acting at other sites. The chain composition of the receptor is alpha 2 beta gamma delta. The two acetylcholine binding sites are on the alpha chains, and two residues contributing to these sites, Cys-192 and Cys-193, have been identified. Furthermore, these adjacent Cys residues are cross-linked by a disulfide bond. In the quaternary structure of the receptor, the chains appear to be arranged in the order alpha gamma alpha beta delta around a central channel. Both the alpha and beta chains contribute to functionally significant NCI binding sites. The addition to receptor-rich membrane from Torpedo electric tissue of agonists (but not competitive antagonists) renders these NCI sites susceptible to photolabeling by the NCI quinacrine azide (QA). Furthermore, this susceptibility is transient, arising in milliseconds and subsiding in hundreds of milliseconds. These transiently susceptible sites are protected by other NCIs against photolabeling by QA. The time-course of the susceptibility and its dependence on agonist-concentration suggest that it might be the transient, rapid-onset desensitized state of the receptor that is most susceptible to photolabeling by QA.

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