Reversible protein kinase C activation in PC12 cells: effect of NGF treatment.

Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+-dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized.

Rapid in vitro conformational changes of the catalytic site of PKC alpha assessed by FIM-1 fluorescence.

To study the activation process of protein kinase C (PKCalpha), we used a fluorescent probe, FIM-1, a bis-indolylmaleimide derivative, which binds to the ATP-binding site on the catalytic domain [Chen, C. S., and Poenie, M. (1993) J. Biol. Chem. 268, 15812]. This enabled us to directly observe the microenvironment of the ATP-binding site in vitro during the activation process. The FIM-1 binding affinity for PKCalpha (EC(50) between 6 and 10 nM) was affected neither by PKCalpha activating conditions nor by enzyme proteolysis. The fluorescence yield of the PKCalpha-FIM-1 complex depended on the PKCalpha activation state. This fluorescence yield was decreased upon proteolysis, which allowed us to study the rate of PKC proteolysis by mu-calpain and its modification by cofactors. Two binding sites were also observed for Ca2+ on the partially activated PKCalpha. After phorbol ester (TPA) application, PKC activation was characterized by biexponential kinetics, including a rapid phase completed within 5 min and a slow phase lasting at least 30 min, which reflected several activation steps. Two different binding sites for TPA were revealed on membrane-associated PKCalpha (EC(50) = 31 +/- 12 and 580 +/- 170 nM), and their modulation by phosphatidylserine and Ca2+ was characterized. The high-affinity TPA binding site was highly conserved, even on the soluble enzyme. Our study shows that binding of low concentrations of TPA triggers conformational changes in the soluble PKCalpha, which affect the microenvironment of its catalytic domain.

Time-resolved imaging of protein kinase C activation during sea urchin egg fertilization.

To study protein kinase C (PKC) activation during sea urchin egg fertilization we used three different fluorescent probes specific for PKC, namely, fim-1, which recognizes the catalytic site of the enzyme, and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. We were able to follow PKC activation during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increased and clustered in the cortical zone of the cell. The process was Ca2+ dependent and was inhibited in the presence of staurosporine, a PKC inhibitor. According to our in vitro probe characterization, this signal increase is due to PKC activation. These findings were further confirmed by Western blot analysis. This initial phase was followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca2(+)-dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization.

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase.

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3-/Cl- exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3-. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.

Initial steps of alpha- and beta-D-glucose binding to intact red cell membrane.

The kinetics of the initial phases of D-glucose binding to the glucose transport protein (GLUT1) of the human red cell can be followed by stopped-flow measurements of the time course of tryptophan (trp) fluorescence enhancement. A number of control experiments have shown that the trp fluorescence kinetics are the result of conformational changes in GLUT1. One shows that nontransportable L-glucose has no kinetic response, in contrast to D-glucose kinetics. Other controls show that D-glucose binding is inhibited by cytochalasin B and by extracellular D-maltose. A typical time course for a transportable sugar, such as D-glucose, consists of a zero-time displacement, too fast for us to measure, followed by three rapid reactions whose exponential time courses have rate constants of 0.5-100 sec-1 at 20 degrees C. It is suggested that the zero-time displacement represents the initial bimolecular ligand/GLUT1 association. Exponential 1 appears to be located at, or near, the external membrane face where it is involved in discriminating among the sugars. Exponential 3 is apparently controlled by events at the cytosolic face. Trp kinetics distinguish the Kd of the epimer, D-galactose, from the Kd for D-glucose, with results in agreement with determinations by other methods. Trp kinetics distinguish between the binding of the alpha- and beta-D-glucose anomers. The exponential 1 activation energy of the beta-anomer, 13.6 +/- 1.4 kcal mol-1, is less than that of alpha-D-glucose, 18.4 +/- 0.8 kcal mol-1, and the two Arrhenius lines cross at approximately 23.5 degrees C. The temperature dependence of the kinetic response following alpha-D-glucose binding illustrates the interplay among the exponentials and the increasing dominance of exponential 2 as the temperature increases from 22.3 to 36.6 degrees C. The existence of these interrelations means that previously acceptable approximations in simplified reaction schemes for sugar transport will now have to be justified on a point-to-point basis.

Conformational changes in human red cell membrane proteins induced by sugar binding.

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

High proton flux through membranes containing antidiuretic hormone water channels.

Antidiuretic hormone (ADH) stimulation of toad urinary bladder granular cells causes simultaneous increases in transepithelial water and H+ permeabilities (PF and PH+, respectively), suggesting that ADH-elicited water channels inserted into granular cell apical membranes might be permeable to both water and H+. We have previously used self-quenching fluorophores entrapped within endocytic vesicles selectively retrieved from water-permeable apical membranes to measure vesicle PF. The membranes of these vesicles possess an extremely high PF such that our measurements provide only minimum estimates of vesicle PF and have limited our ability to quantitate the properties of ADH water channels. We therefore quantitated vesicle PH+ using similar rapid mixing techniques. Vesicle PH+ was 5.1 +/- 0.5 x 10(-3) cm/s. Activation energy of this process was 3.6 +/- 0.6 kcal/mol, indicative of H+ flux through an aqueous channel. The mercurial reagent, para-chloromercuribenzenesulfonate (PCMBS), which inhibits ADH-stimulated transepithelial PF in intact bladders by 50-60%, inhibited vesicle PH+ by 55%. N-Ethylmaleimide and phloretin, which do not alter ADH-stimulated PF, did not affect vesicle PH+. We conclude that membranes containing ADH water channels possess substantial PH+ that likely reflects proton flux through water channels. The apparent high PH+ of the ADH water channel may have important implications for intracellular trafficking of these water channels in ADH-responsive epithelial cells.

Interaction among anion, cation and glucose transport proteins in the human red cell.

The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3.

A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682-1688, 1986) found that C1-/HCO-3 exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4'-diisothiocyano-2,2'-disulfonic stilbene), with a K1 similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4'-dibenzamido-2,2'-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (Ks1 = 93 +/- 24 nM) and a lower affinity site (Ks2 = 430 +/- 260 nM), which are closely similar to values for the red cell of 110 +/- 51 nM for the high-affinity site and 980 +/- 200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). When Cl- replaces citrate in the buffer, the two sites collapse into a single one with Ks1 = 1500 +/- 400 nM, similar to the single Ks1 = 1200 +/- 200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon, J. Membrane Biol. 89:211-223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 microM-1 are characterized by a fast process, tau = 0.14 +/- 0.03 sec, similar to tau = 0.12 +/- 0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell 'band 3' closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.