Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.     

A calcium sensor in the sodium channel modulates cardiac excitability.

Sodium channels are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Calcium ions (Ca2+) have a fundamental role in the coupling of cardiac myocyte excitation and contraction, yet mechanisms whereby intracellular Ca2+ may directly modulate Na channel function have yet to be identified. Here we show that calmodulin (CaM), a ubiquitous Ca2+-sensing protein, binds to the carboxy-terminal 'IQ' domain of the human cardiac Na channel (hH1) in a Ca2+-dependent manner. This binding interaction significantly enhances slow inactivation-a channel-gating process linked to life-threatening idiopathic ventricular arrhythmias. Mutations targeted to the IQ domain disrupted CaM binding and eliminated Ca2+/CaM-dependent slow inactivation, whereas the gating effects of Ca2+/CaM were restored by intracellular application of a peptide modelled after the IQ domain. A naturally occurring mutation (A1924T) in the IQ domain altered hH1 function in a manner characteristic of the Brugada arrhythmia syndrome, but at the same time inhibited slow inactivation induced by Ca2+/CaM, yielding a clinically benign (arrhythmia free) phenotype.     

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.     

Ca2+/calmodulin is critical for brassinosteroid biosynthesis and plant growth

Brassinosteroids are plant-specific steroid hormones that have an important role in coupling environmental factors, especially light, with plant growth and development. How the endogenous brassinosteroids change in response to environmental stimuli is largely unknown. Ca2+/calmodulin has an essential role in sensing and transducing environmental stimuli. Arabidopsis DWARF1 (DWF1) is responsible for an early step in brassinosteroid biosynthesis that converts 24-methylenecholesterol to campesterol. Here we show that DWF1 is a Ca2+/calmodulin-binding protein and this binding is critical for its function. Molecular genetic analysis using site-directed and deletion mutants revealed that loss of calmodulin binding completely abolished the function of DWF1 in planta, whereas partial loss of calmodulin binding resulted in a partial dwarf phenotype in complementation studies. These results provide direct proof that Ca2+/calmodulin-mediated signalling has a critical role in controlling the function of DWF1. Furthermore, we observed that DWF1 orthologues from other plants have a similar Ca2+/calmodulin-binding domain, implying that Ca2+/calmodulin regulation of DWF1 and its homologues is common in plants. These results raise the possibility of producing size-engineered crops by altering the Ca2+/calmodulin-binding property of their DWF1 orthologues.     

Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion

The question concerning whether all membranes fuse according to the same mechanism has yet to be answered satisfactorily. During fusion of model membranes or viruses, membranes dock, the outer membrane leaflets mix (termed hemifusion), and finally the fusion pore opens and the contents mix. Viral fusion proteins consist of a membrane-disturbing 'fusion peptide' and a helical bundle that pin the membranes together. Although SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes form helical bundles with similar topology, it is unknown whether SNARE-dependent fusion events on intracellular membranes proceed through a hemifusion state. Here we identify the first hemifusion state for SNARE-dependent fusion of native membranes, and place it into a sequence of molecular events: formation of helical bundles by SNAREs precedes hemifusion; further progression to pore opening requires additional peptides. Thus, SNARE-dependent fusion may proceed along the same pathway as viral fusion: both use a docking mechanism via helical bundles and additional peptides to destabilize the membrane and efficiently induce lipid mixing. Our results suggest that a common lipidic intermediate may underlie all fusion reactions of lipid bilayers.     

Recruitment of cytosolic proteins to a secretory granule membrane depends on Ca2+-calmodulin

An increase in free calcium triggers catecholamine secretion from chromaffin cells and calmodulin is strongly implicated as the intracellular Ca2+ receptor. In our recent studies of calmodulin action in the chromaffin cell, micromolar Ca2+ concentrations resulted in calmodulin and cytosolic proteins becoming bound to the chromaffin granule membranes. We now report that calmodulin is bound with high affinity to granule membrane proteins of molecular weights (Mrs) 25,000 and 22,000 (25K and 22K) at low Ca2+ (less than 10(-8) M) and to proteins with Mrs 69K and 50K at high Ca2+ (greater than 1 microM). Other cytosolic components (Mrs 70K, 36K, 34K and 32K) require calmodulin for their interfraction with membrane. These proteins separately bound to calmodulin-Sepharose at high Ca2+ concentrations. Although the functions of these adrenal proteins have not been established, the 34K and 32K Mr components co-migrate with clathrin light chains isolated from medullary coated vesicles and the Mr 34K components from both sources share the same one-dimensional peptide map. These interactions were observed at micromolar Ca2+ levels at 'intracellular' conditions of pH and ionic strength and would be expected to occur during secretion from the chromaffin cell.     

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Functional architecture of an intracellular membrane t-SNARE

Lipid bilayer fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, of which one is supplied by the v-SNARE and the other three by the t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and a SNAP-25 protein contributes the other two. Although there are numerous homologues of syntaxin on intracellular membranes, there are only two SNAP-25-related proteins in yeast, Sec9 and Spo20, both of which are localized to the plasma membrane and function in secretion and sporulation, respectively. What replaces SNAP-25 in t-SNAREs of intracellular membranes? Here we show that an intracellular t-SNARE is built from a 'heavy chain' homologous to syntaxin and two separate non-syntaxin 'light chains'. SNAP-25 may thus be the exception rather than the rule, having been derived from genes that encoded separate light chains that fused during evolution to produce a single gene encoding one protein with two helices.     

Clathrin light chains and secretory vesicle binding proteins are distinct

Recently, several groups have initiated studies on cytosolic proteins that bind to isolated secretory vesicle membranes in the presence of Ca2+ in order to identify proteins that may regulate exocytosis. Two major chromaffin granule binding proteins, of molecular weights 32,000 (32K) and 34,000 (34K), were reported to have the same mobility on one-dimensional SDS gels as clathrin-associated light chains from the adrenal medulla, and the 34K granule binding protein the same one-dimensional peptide map as the 34K clathrin light chain. These observations support the hypothesis that Ca2+-dependent recruitment of soluble light chains to the vesicle membrane may nucleate the assembly of a clathrin coat and initiate endocytosis. Here we report that two-dimensional peptide maps of the clathrin light chains and of all chromaffin granule membrane binding proteins in the 30K range are distinct, and therefore fail to support this hypothesis. It has also been suggested that some or all of the vesicle binding proteins require calmodulin for their interaction with the membrane. However, we find that antagonism of calmodulin by trifluoperazine does not prevent the association of the other cytosolic proteins with the chromaffin granule membrane.     

Structural changes of envelope proteins during alphavirus fusion

Alphaviruses are enveloped RNA viruses that have a diameter of about 700?? and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.     

Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate

The actin-binding protein gelsolin requires micromolar concentrations of calcium ions to sever actin filaments, to potentiate its binding to the end of the filament and to promote the polymerization of monomeric actin into filaments. Because transient increases in both intracellular [Ca2+] and actin polymerization accompany the cellular response to certain stimuli, it has been suggested that gelsolin regulates the reversible assembly of actin filaments that accompanies such cellular activations. But other evidence suggests that these activities do not need increased cytoplasmic [Ca2+] and that once actin-gelsolin complexes form in the presence of Ca2+ in vitro, removal of free Ca2+ causes dissociation of only one of two bound actin monomers from gelsolin and the resultant binary complexes cannot sever actin filaments. The finding that cellular gelsolin-actin complexes can be dissociated suggests that a Ca2+-independent regulation of gelsolin also occurs. Here we show that, like the dissociation of profilin-actin complexes, phosphatidylinositol 4,5-bisphosphate, which undergoes rapid turnover during cell stimulation, strongly inhibits the actin filament-severing properties of gelsolin, inhibits less strongly the nucleating ability of this protein and restores the potential for filament-severing activity to gelsolin-actin complexes.     

Differential expression of calmodulin-binding proteins in B, T lymphocytes and thymocytes

Changes in intracellular free Ca2+ are involved in the transmembrane signalling of different cells, including lymphocytes. Since calmodulin (CaM) is a primary receptor for Ca2+ (ref. 4), it may mediate the activation of crucial enzymes after antigen-induced increases in cytosolic Ca2+. Using a biotinylated-CaM (Bio-CaM) detection procedure to identify such proteins, we found that a peptide of relative molecular mass 59,000 (59K) was the predominant soluble CaM-binding protein (CaM-BP) in T cells and B lymphocytes from murine spleen; immunoblotting experiments identified it as a subunit of the CaM-dependent phosphatase, 'calcineurin' (CN). Smaller amounts of larger CaM-BPs, thought to be cytoskeletal-binding proteins, were also detected. CaM-BPs were expressed differentially, with B lymphocytes having four times more of the CN-like protein than T lymphocytes, while in thymocytes, a 65K polypeptide was the major CaM-BP. However, limited proteolysis analysis suggested that this thymus-specific peptide may be a precursor of CN. These data suggest that Ca2+-stimulated protein dephosphorylation may be an important and highly regulated function in lymphoid cells.     

Identification of Na-Ca exchange current in single cardiac myocytes

In cardiac muscle the exchange of intracellular Ca2+ for extracellular Na+ is an important transport mechanism for regulation of the intracellular free Ca2+ concentration [( Ca]i) and hence the contractile strength of the heart. Due to its stoichiometry of greater than or equal to 3:1 Na+/Ca2+ (refs 3,5), Na-Ca exchange is supposed to generate a current across the cell membrane. It is thought that such a current may contribute to cardiac action potential and physiological or pathological pacemaker activity. Although the occurrence of Na-Ca exchange is well documented, a membrane current generated by this transport has not been identified unequivocally. Previous attempts to detect such a current in multicellular preparations, for example, by measuring small current differences after varying the extracellular ionic composition, although providing evidence, did not rule out other possible interpretations. Here we demonstrate that a transient rise in [Ca]i caused by release of Ca from sarcoplasmic reticulum (SR) generates a membrane current in cardiac myocytes. The dependence of this current on the transmembrane gradients for Na+ and Ca2+ and on membrane potential meets the criteria for a current produced by electrogenic Na-Ca exchange. Cyclic activation of this current by release of Ca from the SR can cause maintained spontaneous activity, suggesting that Na-Ca exchange contributes to certain forms of cardiac pacemaking.     

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.