Molecular mechanisms of activation and regulation of ANO1-Encoded Ca2+-Activated Cl- channels.

Ca2+-activated Cl- channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel ß subunits KCNE1/5.

Functional geometry of the permeation pathway of Ca2+-activated Cl-channels inferred from analysis of voltage-dependent block.

We examined the voltage-dependent block of Ca(2+)-activated Cl(-) channels by anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumic acid (NFA) in excised inside-out and outside-out patches from Xenopus oocytes. The fraction of the voltage field (delta) experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from the outside. delta was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for NFA. Because the voltage dependence of the drugs differed, the order of potency was also voltage-dependent. At +100 mV the order of potency was NFA > A9C > DIDS > DPC (K(i) (microm) = 10.1, 18.3, 48, and 111, respectively). Because the drugs are hydrophobic, they can cross the bilayer when applied from the inside and block the channel from the outside. The equilibrium geometries of the blockers were determined by molecular modeling and compared with their blocking positions (delta). This analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 x 0.75 nm, because C(CN)(3)-, which has these dimensions, permeates. The external opening is at least 0.60 x 0.94 nm, because DPC has these dimensions and penetrates the channel approximately 30%.

Anion permeation in Ca(2+)-activated Cl(-) channels.

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.

A hyperpolarization- and acid-activated nonselective cation current in Xenopus oocytes.

Heterologous expression of a variety of membrane proteins in Xenopus oocytes sometimes results in the appearance of a hyperpolarization-activated inward current. The nature of this current remains incompletely understood. Some investigators have suggested that this current is a Cl current, whereas others have identified it as a nonselective cation current. The purpose of this investigation was to characterize this current in more detail. The hyperpolarization-activated inward current (I(IN)) present in native oocytes was composed of a current carried at least partly by Ca and Mg under physiological ionic conditions plus a Ca-activated Cl current. The Ca-activated Cl current was blocked by chelation of cytosolic Ca with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid. When Cl currents were blocked, the cation current could be carried by Ca, Mg, or Co, but not appreciably by Ba, Mn, or Cd. I(IN) was stimulated by intracellular acidification. The properties of I(IN) were quite different from those of the store-operated Ca current. Heterologous expression of transient receptor potential-like gene product (TRPL), one of the members of the transient receptor potential family of putative store-operated Ca channels, apparently resulted in alteration of the voltage sensitivity of the endogenous I(IN).

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals.

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.

Adenophostin A and inositol 1,4,5-trisphosphate differentially activate Cl- currents in Xenopus oocytes because of disparate Ca2+ release kinetics.

Depletion of endoplasmic reticulum Ca2+ stores induces Ca2+ entry from the extracellular space by a process termed "store-operated Ca2+ entry" (SOCE). It has been suggested that the novel fungal metabolite adenophostin-A may be able to stimulate Ca2+ entry without stimulating Ca2+ release from stores. To test this idea further, we compared Ca2+ release, SOCE, and the stimulation of Ca2+-activated Cl- currents in Xenopus oocytes in response to inositol 1,4,5-trisphosphate (IP3) and adenophostin-A injection. IP3 stimulated an outward Cl- current, ICl1-S, in response to Ca2+ release from stores followed by an inward current, ICl2, in response to SOCE. In contrast, low concentrations of adenophostins (AdAs) activated ICl2 without activating ICl1-S, consistent with the suggestion that AdA can activate Ca2+ entry without stimulating Ca2+ release. However, when Ca2+ entry has been stimulated by AdA, Ca2+ stores are largely depleted of Ca2+, as assessed by the inability of ionomycin to release additional Ca2+. The Ca2+ release stimulated by AdA, however, was 7 times slower than the release stimulated by IP3, which could explain the minimal activation of ICl1-S; when Ca2+ is released slowly, the threshold level required for ICl1-S activation is not attained.

Dynamics of calcium regulation of chloride currents in Xenopus oocytes.

Ca-activated Cl currents are widely expressed in many cell types and play diverse and important physiological roles. The Xenopus oocyte is a good model system for studying the regulation of these currents. We previously showed that inositol 1,4,5-trisphosphate (IP3) injection into Xenopus oocytes rapidly elicits a noninactivating outward Cl current (ICl1-S) followed several minutes later by the development of slow inward (ICl2) and transient outward (ICl1-T) Cl currents. In this paper, we investigate whether these three currents are mediated by the same or different Cl channels. Outward Cl currents were more sensitive to Ca than inward Cl currents, as shown by injection of different amounts of Ca or by Ca influx through a heterologously expressed ligand-gated Ca channel, the ionotropic glutamate receptor iGluR3. These data could be explained by two channels with different Ca affinities or one channel with a higher Ca affinity at depolarized potentials. To distinguish between these possibilities, we determined the anion selectivity of the three currents. The anion selectivity sequences for the three currents were the same (I > Br > Cl), but ICl1-S had an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, after consideration of possible errors, the hypothesis that a single type of Cl channel underlies the complex waveforms of the three different macroscopic Ca-activated Cl currents in Xenopus oocytes remains a viable alternative.

Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells.

Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes l-NE transport capacity (Vmax) with little or no change in norepinephrine (NE) Km and without apparent effects on membrane potential monitored directly under current clamp. Over the same time frame, MCh exposure reduces the density of [3H]nisoxetine binding sites (Bmax) in intact cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather than changes in intrinsic transport activity or protein degradation. Similar changes in NE transport and [3H]nisoxetine binding capacity are observed after phorbol ester (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes beta-PMA-mediated effects but produce only a partial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NET activity. In contrast, treatment of cells with the Ca++/ATPase antagonist, thapsigargin in Ca++-free medium, eliminates the staurosporine-insensitive component of MCh regulation. These findings were further corroborated by the ability of [1, 2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester application in Ca++-free medium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric oxide-linked pathways in MCh regulation. Together, these findings 1) provide evidence in support of G-protein coupled receptor-mediated regulation of catecholamine transport, 2) reveal intracellular Ca++-sensitive, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transport evident after mAChR and PKC activation involves a redistribution of NET protein.

Asymmetrical distribution of Ca-activated Cl channels in Xenopus oocytes.

Xenopus oocytes are a popular model system for studying Ca signaling. They endogenously express two kinds of Ca-activated Cl currents, I(Cl-1), and I(Cl-2). I(Cl-1) is activated by Ca released from internal stores and, with appropriate voltage protocols, by Ca influx. In contrast, I(Cl-2) activation is dependent on Ca influx. We are interested in understanding how these two different Cl channels are activated differently by Ca from different sources. One could hypothesize that these channels are activated differently because they are differentially localized near the corresponding Ca source. As an initial investigation of this hypothesis, we examined the distribution of I(Cl-1) and I(Cl-2) channels in the oocyte. We conclude that both I(Cl-1) and (Cl-2) channels are primarily localized to the animal hemisphere of the oocyte, but that capacitative Ca influx occurs over the entire oocyte membrane. Evidence supporting this view includes the following observations: 1) Injection of IP3 into the animal hemisphere produced larger and faster I(Cl-1) responses than injection into the vegetal hemisphere. 2) Exposure of the animal hemisphere to Cl-free solution almost completely abolished I(Cl-1) produced by IP3-induced release of Ca from internal stores or by capacitative Ca entry. 3) Loose macropatch recording showed that both I(Cl-1) and I(Cl-2) currents were approximately four times and approximately three times, respectively, more dense in the animal than in the vegetal hemisphere. 4) Confocal imaging of oocytes loaded with fluorescent Ca-sensitive dyes showed that the time course of activation of I(Cl-1) corresponded to the appearance of the wave of Ca release at the animal pole. 5) Ca release and Ca influx, although twofold higher in the animal pole, were evident over the entire oocyte.

Effects of protein phosphatase and kinase inhibitors on Ca2+ and Cl- currents in guinea pig ventricular myocytes.

It is well-established that in heart, both the L-type Ca2+ channel and the cystic fibrosis transmembrane conductance regulator Cl- channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 microM microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nM for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19-31), and it was not PKA because it was not inhibited by adenosine 3',5'-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl- channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.

Molecular cloning and characterization of an L-epinephrine transporter from sympathetic ganglia of the bullfrog, Rana catesbiana.

Chemical signaling by dopamine (DA) and L-norepinephrine (L-NE) at synapses is terminated by uptake via specialized presynaptic transport proteins encoded by the DA transporter (DAT) and L-NE transporter (NET) genes, respectively. In some vertebrate neurons, particularly the sympathetic neurons of amphibians, L-NE is converted to L-epinephrine (L-Epi, adrenaline) and released as the primary neurotransmitter. Although evidence exists for a molecularly distinct L-Epi transporter (ET) in the vertebrate brain and peripheral nervous system, a transporter specialized for extracellular L-Epi clearance has yet to be identified. To pursue this issue, we cloned transporter cDNAs from bullfrog (Rana catesbiana) paravertebral sympathetic ganglia and characterized functional properties via heterologous expression in non-neuronal cells. A cDNA of 2514 bp (fET) was identified for which the cognate 3.1 kb mRNA is highly enriched in frog sympathetic ganglia. Sequence analysis of the fET cDNA reveals an open reading frame coding for a protein of 630 amino acids. Inferred fET protein sequence bears 75, 66, and 48% amino acid identity with human NET, DAT, and the 5-hydroxytryptamine transporter (SERT), respectively. Transfection of fET confers Na+- and Cl--dependent catecholamine uptake in HeLa cells. Uptake of [3H]-L-NE by fET is inhibited by catecholamines in a stereospecific manner. L-Epi and DA inhibit fET-mediated [3H]-L-NE uptake more potently than they inhibit [3H]-L-NE uptake by human NET (hNET), whereas L-NE exhibits equivalent potency between the two carriers. Moreover, fET exhibits a greater maximal velocity (Vmax) for the terminal products of catecholamine biosynthesis (L-Epi > L-NE >> DA), unlike hNET, in which a Vmax rank order of L-NE > DA > L-Epi is observed. fET-mediated transport of catecholamines is sensitive to cocaine and tricyclic antidepressants, with antagonist potencies significantly correlated with hNET inhibitor sensitivity. Amino acid conservation and divergence of fET with mammalian catecholamine transporters help define residues likely to be involved in catecholamine recognition and translocation as well as blockade by selective reuptake inhibitors.

Effects of adenophostin-A and inositol-1,4,5-trisphosphate on Cl- currents in Xenopus laevis oocytes.

Adenophostin-A, a novel compound isolated from cultures of Penicillium brevicompactum, has been shown to stimulate Ca2+ release from inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores in microsomal preparations, permeabilized cells, and lipid vesicles containing purified IP3 receptor. The purpose of the current study was to compare the effects of adenophostin-A and IP3 on Ca2+ release from stores and Ca2+ influx in intact Xenopus laevis oocytes. Ca2+ influx though store-operated Ca2+ channels and Ca2+ release from stores were monitored by measuring two Ca2+ -activated Cl- currents that can be used as real-time indicators of Ca2+ release and Ca2+ influx (I(Cl-1) and I(Cl-2), respectively). We find that high concentrations (final intraoocyte concentrations of 5-10 microM) of adenophostin-A and IP3 stimulate a large Ca2+ release from stores (as measured by I(Cl-1)) followed by Ca2+ influx (as measured by I(Cl-2)). Low concentrations (approximately 50 nM) of IP3 stimulate oscillations in Ca2+ release without stimulating Ca2+ influx. In contrast, low concentrations of adenophostin-A can stimulate Ca2+ influx without stimulating a large Ca2+ release. However, Ca2+ influx did not occur in the complete absence of Ca2+ release. Therefore, it is unlikely that adenophostin-A directly stimulates store-operated Ca2+ channels. We hypothesize that adenophostin-A releases Ca2+ from a subpopulation of stores that is tightly coupled to store-operated Ca2+ channels.

Activation of different Cl currents in Xenopus oocytes by Ca liberated from stores and by capacitative Ca influx.

Xenopus oocytes are an excellent model system for studying Ca signaling. The purpose of this study was to characterize in detail the Ca-activated Cl currents evoked by injection of inositol 1,4,5-trisphosphate (IP3) into Xenopus oocytes voltage-clamped with two microelectrodes. Injection of IP3 into Xenopus oocytes activates two different Ca-activated Cl currents. ICl-1 is stimulated rapidly (within 5 s after IP3 injection), exhibits time-dependent activation upon depolarization, a linear instantaneous IV relationship with a reversal potential near ECl, and a curvilinear activation curve with an approximate half-maximal activation voltage of > 200 mV. ICl-2D is stimulated slowly after IP3 injection (half-maximal stimulation occurs approximately 3 min after injection). ICl-2D has a strongly outwardly rectifying instantaneous IV relationship with a reversal potential near ECl and is activated by hyperpolarization with a half-maximal activation voltage of -105 mV. ICl-2D cannot be activated by Ca released from stores but is activated by Ca influx. In contrast, ICl-1 can be stimulated by Ca released from intracellular Ca stores. It can also be stimulated by Ca influx through store-operated channels if the Ca driving force is increased by a hyperpolarization immediately before the depolarization that gates ICl-1 channels. The description of two currents activated by influx and Ca release from stores provides new insights into and questions about the regulation of Ca in Xenopus oocytes.

Calphostin C, a widely used protein kinase C inhibitor, directly and potently blocks L-type Ca channels.

Calphostin C is a widely used inhibitor of protein kinase C; in the past 4 years at least 350 articles have been published using this drug as a selective inhibitor of protein kinase C. In this paper, we show that calphostin C also potently inhibits cardiac L-type Ca channels by a mechanism that does not involve changes in adenosine 3',5'-cyclic monophosphate levels or dephosphorylation. The inhibition requires illumination by visible light during exposure to calphostin C. The Ca current (ICa) that remains after partial inhibition of ICa has the same voltage-dependent characteristics as the control current.

Effect of arachidonic acid on the L-type calcium current in frog cardiac myocytes.

1. External application of the unsaturated fatty acid arachidonic acid (AA) to frog ventricular cells caused a large inhibition (approximately 85%) of the L-type calcium current (ICa,L) previously stimulated by the beta-adrenergic agonist isoprenaline (Iso). The concentration producing half-maximal inhibition (K1/2) was 1.52 microM. The inhibitory effect did not affect the peak current-voltage relationship but produced a negative shift in the inactivation curve. 2. The inhibitory effect of AA also occurred in cells internally perfused with cAMP and non-hydrolysable analogues of cAMP. These data suggest that AA is acting by a mechanism located beyond adenylyl cyclase and does not involve changes in intracellular cAMP levels. 3. AA also inhibited the calcium current stimulated by internal perfusion with the catalytic subunit of protein kinase A (PKA), suggesting that AA acts downstream of channel phosphorylation. 4. The inhibitory effect of AA on the isoprenaline- or cAMP-stimulated ICa,L is largely reduced in cells internally perfused with the thiophosphate donor analogue of ATP, ATP gamma S, or protein phosphatase 1 and 2A inhibitors like microcystin (MC) or okadaic acid (OA). External application of the phosphatase inhibitor calyculin (Caly) also reduced the AA effect. These data suggested that the AA effect on ICa,L involves activation of protein phosphatase activity. 5. The effect of AA on ICa,L was not affected by staurosporine, an inhibitor of protein kinases. It was also unaffected in cells internally perfused with GTP gamma S. These results suggest that neither a PKC- nor a G-protein-mediated mechanism are likely to be involved in the effect of AA on ICa,L. 6. A saturated fatty acid, myristic acid (MA), had no inhibitory effect on the isoprenaline-stimulated Ca2+ current, whereas, in the same cells arachidonic acid produced approximately 85% inhibition of ICa,L. 7. The inhibitory effect of AA was not affected by exposing the cells to indomethacin (Indo), an inhibitor of the metabolism of AA by cyclo-oxygenase, nor nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway. However, the non-metabolizable analogue of AA, 5,8,11,14-eicosatetraynoic acid (ETYA), was without effect on the isoprenaline-stimulated ICa,L. 8. These results suggest that AA inhibits ICa,L via a mechanism which involves, in part, stimulation of protein phosphatase activity. This process could provide a new mechanism in the modulation of calcium channel activity.

Nitric oxide synthase does not participate in negative inotropic effect of acetylcholine in frog heart.

In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.

Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase.

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10-30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.

Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells.

We have studied the postnatal changes in the levels of isoforms of stimulatory (Gs) and inhibitory (Gi) G-proteins, cAMP and cGMP in washed particulate membranes (WPM) from whole ventricles as well as from isolated ventricular myocytes and have also measured adenylyl cyclase (AC) activity in WPM prepared from isolated myocytes of adult (AD) and newborn (NB) rabbit heart. Immunoblot analysis for the levels of Gi alpha 1, G alpha 2, Gi alpha 3 and Gs alpha subunits showed that Gi alpha 2 and Gi alpha 3 were higher in WPM from whole ventricles of NB compared to AD. This ratio was much higher in WPM from isolated ventricular myocytes since Gi alpha 2 and Gi alpha 3 were either absent or present in extremely low immunodetectable levels in WPM from AD ventricular myocytes. Gi alpha 1 levels were not different for AD compared to NB WPM, whether prepared from whole ventricle or from isolated myocytes. Two forms of Gs alpha, a small form (Gs alpha-S) and a large form (Gs alpha-L), were immunodetected at 43 and 48 kDa, respectively. The Gs alpha-S form was higher in AD WPM and the Gs alpha-L form was higher in NB WPM while the total Gs alpha(L+S) was not different. The Gs alpha results for WPM from isolated myocytes were not different from the results for WPM from whole ventricles. Basal levels of cAMP were 80% higher in NB compared to AD whole ventricles and were 200% higher in NB compared to AD isolated myocytes. Levels of cGMP were 4-5 fold higher in NB than in AD myocytes and ventricular tissue. Basal AC activity was higher in NB than in AD WPM from isolated myocytes and was enhanced by Gpp(NH)p pretreatment in AD but not in NB WPM. The isoproterenol-induced increase in AC activity was higher in AD compared to NB WPM and was completely abolished by Gpp(NH)p pretreatment in NB but not in AD WPM. Forskolin caused a greater increase in AC activity in NB than in AD WPM. The post-natal decrease in the levels of Gi alpha 2 and Gi alpha 3, particularly in isolated ventricular myocytes, may help to explain the smaller effects of isoproterenol and greater muscarinic influence on ICa, as we previously showed, and the smaller effect of isoproterenol on AC activity in NB compared to AD WPM.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular physiology of norepinephrine and serotonin transporters.

Cocaine- and antidepressant-sensitive norepinephrine and serotonin transporters (NETs and SERTs) are closely related members of the Na+/Cl- transporter gene family, whose other members include transporters for inhibitory amino acid transmitters, neuromodulators, osmolytes and nutrients. Availability of cloned NET and SERT cDNAs has permitted rapid progress in the definition of cellular sites of gene expression, the generation of transporter-specific antibodies suitable for biosynthetic and localization studies, the examination of structure-function relationships in heterologous expression systems and a biophysical analysis of transporter function. In situ hybridization and immunocytochemical studies indicate a primary expression of NET and SERT genes in brain by noradrenergic and serotonergic neurons, respectively. Both NET and SERT are synthesized as glycoproteins, with multiple glycosylation states apparent for SERT proteins in the brain and periphery. N-glycosylation of NET and SERT appears to be essential for transporter assembly and surface expression, but not for antagonist binding affinity. Homology cloning efforts have revealed novel NET and SERT homologs in nonmammalian species that are of potential value in the delineation of the precise sites for substrate and antagonist recognition, including a Drosophila melanogaster SERT with NET-like pharmacology. Electrophysiological recording of human NETs and SERTs stably expressed in HEK-293 cells reveals that both transporters move charge across the plasma membrane following the addition of substrates; these currents can be blocked by NET-and SERT-selective antagonists as well as by cocaine.

Dissection of eukaryotic transmembrane signalling using Chlamydomonas.

Novel insights and surprises are often generated when investigators choose an organism that permits a new approach to a problem. For example, secretory and cell-cycle mutants in yeast have provided quantum leaps in elucidating these processes. Similarly, genetic systems are providing exciting new insights into signal transduction. The 'green yeast' Chlamydomonas has the potential to be a particularly rich organism for genetic analysis of signal transduction because, although unicellular, it has several interesting behaviours, which are discussed in this article by Lynne Quarmby and Criss Hartzell. Phototaxis results from the transduction of a light signal received by the eyespot to changes in flagellar beat. The mating reactions, which culminate in the fusion of gametes, are initiated in response to adhesion of flagellar proteins. Deflagellation, or flagellar shedding, is an acute response to a variety of stimuli. Molecular genetic analysis of behavioural mutants is providing new directions for understanding signal integration and segregation.