Genetic Analysis of Mutants with a Reduced Ca2+-Dependent K+ Current in PARAMECIUM TETRAURELIA

Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.     

Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin

TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca(2+) influx in a variety of epithelial cells. The activity of these Ca(2+) channels is tightly controlled by the intracellular Ca(2+) concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca(2+)-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca(2+) sensor mediating the regulation of channel activity was investigated. Overexpression of Ca(2+)-insensitive CaM mutants (CaM(1234) and CaM(34)) significantly reduced the Ca(2+) as well as the Na(+) current of TRPV6- but not that of TRPV5-expressing HEK293 cells. By combining pull-down assays and co-immunoprecipitations, we demonstrated that CaM binds to both TRPV5 and TRPV6 in a Ca(2+)-dependent fashion. The binding of CaM to TRPV6 was localized to the transmembrane domain (TRPV6(327-577)) and consensus CaM-binding motifs located in the N (1-5-10 motif, TRPV6(88-97)) and C termini (1-8-14 motif, TRPV6(643-656)), suggesting a mechanism of regulation involving multiple interaction sites. Subsequently, chimeric TRPV6/TRPV5 proteins, in which the N and/or C termini of TRPV6 were substituted by that of TRPV5, were co-expressed with CaM(34) in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM(34)-induced reduction of the Ca(2+) and Na(+) currents. These results suggest that CaM positively affects TRPV6 activity upon Ca(2+) binding to EF-hands 3 and 4, located in the high Ca(2+) affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.     

Calmodulin mediates Ca2+ sensitivity of sodium channels

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.     

Genetic dissection of Ca2(+)-dependent ion channel function in Paramecium.

The ciliated protozoan, Paramecium, broadcasts the activity of its individual ion channel classes through its swimming behaviour. This fact has made it possible to isolate mutants with defective ion currents, simply by selecting individuals with abnormal swimming patterns. At least four of Paramecium's ion currents are activated by rising intracellular calcium concentration, including two K+ currents and a Na+ current. A variety of cell lines with defects in these Ca2(+)-dependent currents have been isolated: in several cases, the defects have been traced to mutations in the structural gene for calmodulin. Sequence analysis of calmodulins from these and other Ca2(+)-dependent ion-current mutants may enable a detailed mapping of putative channel interaction domains on the surface of the calmodulin molecule.     

Ruthenium red inhibits the binding of calcium to calmodulin required for enzyme activation.

The Ca(2+)-calmodulin (CaM)-dependent activation of myosin light chain kinase is inhibited by ruthenium red competitively with respect to Ca2+, with a Ki value of 8.6 microM. The binding of Ca2+ to CaM is inhibited by micromolar concentrations of ruthenium red. In the absence of Ca2+, CaM has two binding sites for ruthenium red with the dissociation constants of 0.36 and 8.7 microM, respectively. Ca2+ antagonizes the binding of ruthenium red to the low-affinity site on CaM. Binding of ruthenium red to the high-affinity site is not affected by Ca2+. The low- and high-affinity sites for ruthenium red are shown to be located in the NH2-terminal half and the COOH-terminal half of CaM, respectively. Lower concentrations of ruthenium red are needed for enzyme inactivation than for the dissociation of enzyme-CaM-Sepharose complex, suggesting these events have different Ca2+ requirements. Moreover, ruthenium red inhibits Ca(2+)-induced contraction of depolarized vascular smooth muscle in a competitive manner with respect to Ca2+. These results suggest that ruthenium red may be a new type of CaM antagonist that inhibits the binding of Ca2+ to CaM and thereby inhibits Ca(2+)-CaM-dependent enzymes and smooth muscle contraction competitively with respect to Ca2+.     

Potassium currents in presynaptic hair cells of Hermissenda.

Apart from their primary function as balance sensors, Hermissenda hair cells are presynaptic neurons involved in the Ca(2+)-dependent neuronal plasticity in postsynaptic B photoreceptors that accompanies classical conditioning. With a view to beginning to understand presynaptic mechanisms of plasticity in the vestibulo-visual system, a locus for conditioning-induced neuronal plasticity, outward currents that may govern the excitability of hair cells were recorded by means of a whole-cell patch-clamp technique. Three K+ currents were characterized: a 4-aminopyridine-sensitive transient outward K+ current (IA), a tetraethyl ammonium-sensitive delayed rectifier K+ current (IK,V), and a Ca(2+)-activated K+ current (IK,Ca). IA activates and decays rapidly; the steady-state activation and inactivation curves of the current reveal a window current close to the apparent resting voltage of the hair cells, suggesting that the current is partially activated at rest. By modulating firing frequency and perhaps damping membrane oscillations, IA may regulate synaptic release at baseline. In contrast, IK,V and IK,Ca have slow onset and exhibit little or no inactivation. These two K+ currents may determine the duration of the repolarization phase of hair-cell action potentials and hence synaptic release via Ca2+ influx through voltage-gated Ca2+ channels. In addition, IK,Ca may be responsible for the afterhyperpolarization of hair cell membrane voltage following prolonged stimulation.     

Molecular mechanism for divergent regulation of Cav1.2 Ca2+ channels by calmodulin and Ca2+-binding protein-1

Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.     

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.     

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating

Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.     

Calcium current activated upon hyperpolarization of Paramecium tetraurelia

Hyperpolarization of Paramecium tetraurelia under conditions where K+ currents are suppressed elicits an inward current that activates rapidly toward a peak at 25-80 ms and decays thereafter. This peak current (Ihyp) is not affected by removing Cl ions from the microelectrodes used to clamp membrane potential, or by changing extracellular Cl- concentration, but is lost upon removing extracellular Ca2+. Ihyp is also lost upon replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Co2+, Mg2+, Mn2+, or Sr2+, suggesting that the permeability mechanism that mediates Ihyp is highly selective for Ca2+. Divalent cations also inhibit Ihyp when introduced extracellularly, in a concentration- and voltage-dependent manner. Ba2+ inhibits Ihyp with an apparent dissociation constant of 81 microM at -110 mV, and with an effective valence of 0.42. Ihyp is also inhibited reversibly by amiloride, with a dissociation constant of 0.4 mM. Ihyp is not affected significantly by changes in extracellular Na+, K+, or H+ concentration, or by EGTA injection. Also, it is unaffected by manipulations or mutations that suppress the depolarization-activated Ca2+ current or the various Ca(2+)-dependent currents of Paramecium. We suggest that Ihyp is mediated by a novel, hyperpolarization-activated calcium conductance that is distinct from the one activated by depolarization.     

Elementary mechanisms producing facilitation of Cav2.1 (P/Q-type) channels

The regulation of Ca(V)2.1 (P/Q-type) channels by calmodulin (CaM) showcases the powerful Ca(2+) decoding capabilities of CaM in complex with the family of Ca(V)1-2 Ca(2+) channels. Throughout this family, CaM does not simply exert a binary on/off regulatory effect; rather, Ca(2+) binding to either the C- or N-terminal lobe of CaM alone can selectively trigger a distinct form of channel modulation. Additionally, Ca(2+) binding to the C-terminal lobe triggers regulation that appears preferentially responsive to local Ca(2+) influx through the channel to which CaM is attached (local Ca(2+) preference), whereas Ca(2+) binding to the N-terminal lobe triggers modulation that favors activation via Ca(2+) entry through channels at a distance (global Ca(2+) preference). Ca(V)2.1 channels fully exemplify these features; Ca(2+) binding to the C-terminal lobe induces Ca(2+)-dependent facilitation of opening (CDF), whereas the N-terminal lobe yields Ca(2+)-dependent inactivation of opening (CDI). In mitigation of these interesting indications, support for this local/global Ca(2+) selectivity has been based upon indirect inferences from macroscopic recordings of numerous channels. Nagging uncertainty has also remained as to whether CDF represents a relief of basal inhibition of channel open probability (P(o)) in the presence of external Ca(2+), or an actual enhancement of P(o) over a normal baseline seen with Ba(2+) as the charge carrier. To address these issues, we undertake the first extensive single-channel analysis of Ca(V)2.1 channels with Ca(2+) as charge carrier. A key outcome is that CDF persists at this level, while CDI is entirely lacking. This result directly upholds the local/global Ca(2+) preference of the lobes of CaM, because only a local (but not global) Ca(2+) signal is here present. Furthermore, direct single-channel determinations of P(o) and kinetic simulations demonstrate that CDF represents a genuine enhancement of open probability, without appreciable change of activation kinetics. This enhanced-opening mechanism suggests that the CDF evoked during action-potential trains would produce not only larger, but longer-lasting Ca(2+) responses, an outcome with potential ramifications for short-term synaptic plasticity.     

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.     

Identification of the components controlling inactivation of voltage-gated Ca2+ channels

Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker.     

Potassium current inhibition by nonselective cation channel-mediated sodium entry in rat pheochromocytoma (PC-12) cells.

Under physiological conditions, nonselective cation (NSC) channels mediate the entry of cations into cells, the most important being Na+ and Ca2+. In contrast to the Ca(2+)-dependent signaling mechanisms, little is known about the consequences and the spatial distribution of intracellular [Na+] elevation. In this study we demonstrate that Na+ entry, during the opening of ATP-activated NSC channels, leads to an inhibition of voltage-dependent K+ currents (IK) in cromaffin-like undifferentiated PC-12 cells. The effect was dependent on the charge carrier as well as on the density of the ATP-activated current. Extracellular alkali cations (Na+, Li+) were more efficient than NH4+ in suppressing IK. Intracellular infusion of Na+ had the same effect as Na+ influx through ATP-activated NSC channels. The inhibition of IK persisted when the total ATP-induced Na+ entry was reduced by membrane depolarization, suggesting a spatial restriction of the required Na+ accumulation. Our results indicate that NSC channels influence the function of other ion channels by changing local intracellular ion concentrations.