Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 2. Effects of phosphorylation by cAMP-dependent protein kinase.

The effects of phosphorylation on the voltage-dependent properties of dihydropyridine-sensitive Ca channels of skeletal muscle were studied. Single channel currents were recorded upon incorporation of transverse tubule membranes into planar bilayers that were kept polarized at near physiological resting potential and subjected to depolarizing pulses under voltage clamp. Studies were conducted to analyze the properties of the channels at both the single channel and macroscopic level, using methods introduced in the preceding paper (Ma et al., 1991. Biophys. J. 60: 890-901.). Addition of the catalytic subunit of cAMP-dependent protein kinase to the cis (intracellular) side of the bilayers containing channels resulted in: (a) an increase in open channel probability at all voltages above -50 mV; (b) a leftward shift (by 7 mV) in the curve describing the voltage-dependence of activation; (c) an approximate twofold decrease in the rate of inactivation; and (d) an increase in the availability of the channel. These findings provide new insights at the single channel level into the mechanism of modulation of the dihydropyridine-sensitive Ca channels of skeletal muscle by signal transduction events that involve elevation in cAMP and activation of the cAMP-dependent protein kinase.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.     

Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltage-dependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating models of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.     

Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 1. Kinetics and voltage dependence of gating.

Rabbit skeletal muscle transverse tubule (T) membranes were fused with planar bilayers. Ca channel activity was studied with a "cellular" approach, using solutions that were closer to physiological than in previous studies, including asymmetric extracellular divalent ions as current carriers. The bilayer was kept polarized at -80 mV and depolarizing pulses were applied under voltage clamp. Upon depolarization the channels opened in a steeply voltage-dependent manner, and closed rapidly at the end of the pulses. The activity was characterized at the single-channel level and on macroscopic ensemble averages of test-minus-control records, using as controls the null sweeps. The open channel events had one predominant current corresponding to a conductance of 9 pS (100 mM Ba2+). The open time histogram was fitted with two exponentials, with time constants of 5.8 and 30 ms (23 degrees C). Both types of events were virtually absent at -80 mV. The average open probability (fractional open time) increased sigmoidally from 0 to a saturation level of 0.08, following a Boltzmann function centered at -25 mV and with a steepness factor of 7 mV. Ensemble averages of test-minus-control currents showed a sigmoidal activation followed by inactivation during the pulse and deactivation (closing) after the pulse. The ON time course was well fitted with "m3h" kinetics, with tau m = 120 ms and tau h = 1.2 s. Deactivation was exponential with tau = 8 ms. This study demonstrates a technique for obtaining Ca channel events in lipid bilayers that are strictly voltage dependent and exhibit most of the features of the macroscopic ICa. The technique provides a useful approach for further characterization of channel properties, as exemplified in the accompanying paper, that describes the consequences on channel properties of phosphorylation by cAMP dependent protein kinase.     

Ca channel gating during cardiac action potentials.

How do Ca channels conduct Ca ions during the cardiac action potential? We attempt to answer this question by applying a two-microelectrode technique, previously used for Na and K currents, in which we record the patch current and the action potential at the same time (Mazzanti, M., and L. J. DeFelice. 1987. Biophys. J. 12:95-100, and 1988. Biophys. J. 54:1139-1148; Wellis, D., L. J. DeFelice, and M. Mazzanti. 1990. Biophys. J. 57:41-48). In this paper, we also compare the action currents obtained by the technique with the step-protocol currents obtained during standard voltage-clamp experiments. Individual Ca channels were measured in 10 mM Ca/1 Ba and 10 mM Ba. To describe part of our results, we use the nomenclature introduced by Hess, P., J. B. Lansman, and R. W. Tsien (1984. Nature (Lond.). 311:538-544). With Ba as the charge carrier, Ca channel kinetics convert rapidly from long to short open times as the patch voltage changes from 20 to -20 mV. This voltage-dependent conversion occurs during action potentials and in step-protocol experiments. With Ca as the charge carrier, the currents are brief at all voltages, and it is difficult to define either the number of channels in the patch or the conductance of the individual channels. Occasionally, however, Ca-conducting channels spontaneously convert to long-open-time kinetics (in Hess et al., 1984, notation, mode 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.     

Molecular mechanism of calcium channel block by isradipine. Role of a drug-induced inactivated channel conformation

The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.     

Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.     

Biochemical status of renal epithelial Na+ channels determines apparent channel conductance, ion selectivity, and amiloride sensitivity.

Purified bovine renal papillary Na+ channels, when reconstituted into planar lipid bilayers, reside in three conductance states: a 40-pS main state, and two subconductive states (12-13 pS and 24-26 pS). The activity of these channels is regulated by phosphorylation and by G-proteins. Protein kinase A (PKA)-induced phosphorylation increased channel activity by increasing the open state time constants from 160 +/- 30 (main conductance), and 15 +/- 5 ms (both lower conductances), respectively, to 365 +/- 30 ms for all of them. PKA phosphorylation also altered the closed time of the channel from 250 +/- 30 ms to 200 +/- 35 ms, thus shifting the channel into a lower-conductance, long open time mode. PKA phosphorylation increased the PNa:PK of the channel from 7:1 to 20:1, and shifted the amiloride inhibition curve to the right (apparent K(i)amil from 0.7 to 20 microM). Pertussis toxin-induced ADP-ribosylation of either phosphorylated of either phosphorylated or nonphosphorylated channels decreased the PNa:PK to 2:1 and 4:1, respectively, and altered K(i)amil to 8 and 2 microM for phosphorylated and nonphosphorylated channels, respectively. GTP-gamma-S treatment of either phosphorylated or nonphosphorylated channels resulted in an increase of PNa:PK to 30:1 and 10:1, respectively, and produced a leftward shift in the amiloride dose-response curve, altering K(i)amil to 0.5 and 0.1 microM, respectively. These results suggest that amiloride-sensitive renal Na+ channel biophysical characteristics are not static, but depend upon the biochemical state of the channel protein and/or its associated G-protein.     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.     

Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of lens fiber membranes

Summary Major intrinsic polypeptide (MIP), a 28-kDa protein isolated from lens fiber cell membranes, forms large, nonselective channels when reconstituted into lipid bilayers. MIP channels are regulated by voltage, such that these channels close when the potential across the membrane is greater than 30 mV. We have investigated the modulation of the voltage-dependent closure of MIP channels by phosphorylation. In this report, we describe the isolation of two isomers of MIP from lens fiber cell membranes. These isomers differ by a single phosphate at a protein kinase A phosphorylation site. The phosphorylated isomer produces channels that close in response to applied voltages when reconstituted into bilayers. The nonphosphorylated isomer produces voltage-independent hannels. Direct phosphorylation with protein kinase A converts voltage-independent channels to voltage-dependent channels in situ. Analyses of macroscopic and single channel currents suggest that phosphorylation increases the voltage-dependent closure of MIP channels by increasing closed channel lifetimes and the rate of channel closure following the application of voltage.The authors gratefully acknowledge the gift of the monoclonal antibody to MIP from Drs. David Paul and Dan Goodenough. We thank Dr. Irwin Levitan for the kind gift of purified protein kinase A catalytic subunit. We also thank Ms. Mary Hawley for invaluable technical support and Mr. Paul Ross for help in generating Fig. 10. This work was supported, in part, by NIH grants EY04110 and EY05661 and a NEI postdoctoral fellowship to GRE.     

Fluorescent probes of electrostatic potential 1 nm from the membrane surface

We measured the electrostatic potential 1 nm from the surface of charged phospholipid bilayer membranes to test the predictions of the Gouy-Chapman theory. Fluorescent probes (anthraniloyl, 5-(dimethylamino)naphthalene-1-sulfonyl, Lucifer yellow) were attached covalently to the sialic acid residue of the ganglioside galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylc eramide (GM1). These fluorescent gangliosides were incorporated into neutral [phosphatidylcholine (PC)] or charged [phosphatidylserine (PS)] phospholipid bilayers, and the fluorescence was quenched with the cations thallium and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (tempamine). We calculated the electrostatic potential at the chromophore from the quenching ratio using the Boltzmann relation: the average potential was -30 mV for PS bilayers in 0.1 M NaNO3. We assume the chromophore is 1 nm from the surface because X-ray diffraction measurements demonstrate that the sialic acid residue of GM1 is 1 nm from the surface of a PC/GM1 bilayer [McDaniel, R. V., & McIntosh, T. J. (1986) Biophys. J. 49, 94-96]. We also used thallium and tempamine to quench the fluorescence of chromophores located at the surface of the PS membranes; in 0.1 M NaNO3 the average surface potential was -80 mV, which agrees with other measurements. The Gouy-Chapman theory predicts that the potential 1 nm from a membrane with a surface potential of -80 mV is -24 mV; this prediction agrees qualitatively with the experimental results obtained with fluorescent gangliosides.     

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.     

Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.

We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions.     

Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.     

Single potassium channels with delayed rectifier behavior from lobster axon membranes.

Single-channel potassium currents from lobster axon membranes were studied in planar bilayers made from monolayers. Channel-opening events are grouped by time, forming bursts with an average duration of 4.5 ms. The mean open time at 0 mV is 1.8 ms. The frequency of bursts is voltage dependent, increasing e-fold per 12-16 mV. At sufficiently high positive voltages, channels inactivate. Measured from reversal potentials, channels discriminate against Na+ by a permeability ratio PNa/PK of 1:30. The channel is blocked by tetraethylammonium and nonyltrimethylammonium in a voltage-dependent manner and at concentrations similar to those used in whole-axon experiments. Voltage-dependent block by Cs+ suggests that more than one ion may occupy the channel simultaneously. The kinetics and selectivity of this channel suggest that purified axolemma contains active K+ channels that are likely to participate in delayed rectification in the lobster axon membrane.     

Atrial natriuretic factor inhibits autophosphorylation of protein kinase C and A 240-kDa protein in plasma membranes of bovine adrenal glomerulose cells: Involvement of cGMP-dependent and independent signal transduction mechanisms

To clarify the intracellular signalling mechanisms of atrial natriuretic factor (ANF), we studied its effect on protein phosphorylation in plasma membranes of bovine adrenal cortical cells. ANF (1×10^–7 M) inhibited phosphorylation of the 78-kDa protein kinase C (PKC) and a 240-kDa protein in specific manner. In parallel experiments, cGMP (0.5 mM) inhibited phosphorylation of only the 78-kDa PKC but it did not affect phosphorylation of the 240-kDa protein. Phosphorylation of the 78-kDa PKC was enhanced in a Ca²⁺-/phospholipid-dependent manner. However, after prolonged preincubation of plasma membranes with Ca²⁺ (0.5 mM), the incorporation of³²P-radioactivity rapidly decreased in the 78-kDa PKC and subsequently increased in the 45- and 48-kDa protein bands due to Ca²⁺-dependent proteolytic degradation of 78-kDa PKC. Polyclonal antibodies against brain PCK were used to immunoblot and immunoprecipitate the 78-kDa PKC. Preincubation of plasma membranes with Ca²⁺ for varying times, followed by immunoblotting revealed a gradual loss of the immunoreactive 78-kDa PKC band in a time-dependent manner. Immunoprecipitation of phosphorylated 78-kDa PKC in plasma membranes showed that its phosphorylation was significantly inhibited in the presence of ANF as compared to control membranes, phosphorylated in the absence of ANF. The results in this present study document a new signal transduction mechanism of ANF at molecular level which possibly involves dephosphorylation of the 78-kDa PKC and a 240-kDa protein in a cGMP-dependent and-independent manner in bovine adrenal glomerulosa cell membranes. (Mol Cell Biochem141: 103–111, 1994)     

The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. Evidence for direct regulation independent of phosphorylation by cAMP-dependent protein kinase or stimulation by a dihydropyridine agonist

We demonstrated recently that purified preparations of Gs, the stimulatory G protein of adenylyl cyclase, can stabilize Ca2+ channels in inside-out cardiac ventricle membrane patches stimulated prior to excision by the beta-adrenergic agonist isoprenaline or by the dihydropyridine agonist Bay K 8644 and that such preparations of Gs can restore activity to spontaneously inactivated cardiac Ca2+ channels incorporated into planar lipid bilayers (Yatani, A., Codina, J., Reeves, J.P., Birnbaumer, L., and Brown, A.M. (1987) Science 238, 1288-1292). To test whether these effects represented true stimulation and to further identify the G protein responsible, we incorporated skeletal muscle T-tubule membranes into lipid bilayers and studied the response of their Ca2+ channels to G proteins, specifically Gs, and manipulations known to be specific for Gs. In contrast to cardiac channels, incorporated T-tubule Ca2+ channels exhibit stable average activities over prolonged periods of time (up to 20 min at room temperature), allowing assessment of possible effects of G proteins under steady-state assay conditions. We report that exogenously added human erythrocyte GTP gamma S (guanosine 5'-O-(3-thiotriphosphate]-activated Gs (Gs) or its resolved GTP gamma S-activated alpha subunit (alpha s) stimulate T-tubule Ca2+ channels by factors of 2-3 in the presence of Bay K 8644, and of 10-20 in the absence of Bay K 8644 and that they do so in a manner that is independent of concurrent or previous phosphorylation by cAMP-dependent protein kinase. Activation of purified Gs by cholera toxin increases both its adenylyl cyclase stimulatory and its Ca2+ channel stimulatory effects. Ca2+ channels previously stimulated by the combined actions of Bay K 8644 and cAMP-dependent protein kinase still respond to Gs. We conclude that the responses seen are due to Gs rather than a contaminant, that the effect on Ca2+ channel activity is that of a true stimulation, akin to that on adenylyl cyclase, and show that a given G protein may regulate more than one effector system.     

Fast-deactivating calcium channels in chick sensory neurons

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.