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D W Hilgemann

Publications and source records attributed to D W Hilgemann.

At least 37 records · Page 2Linked to original sources
PubMed1995

Modulation of Na+,Ca2+ exchange current by EGTA calcium buffering in giant cardiac membrane patches.

Effects of calcium buffering by EGTA were examined on sodium-calcium exchange currents (INaCa) in inside-out giant cardiac membrane patches. Free calcium concentrations (Ca2+) were monitored with a calcium electrode and a fluorescent calcium indicator (Calcium Green-5N). With 1.8 microM cytoplasmic Ca2+, inward INaCa increased 2-fold at -120 mV when EGTA concentration was increased from 0.1 mM to 10 mM (37 degrees C and 140 mM extracellular sodium). Stimulation by EGTA was decreased or abolished under conditions of attenuated exchanger turnover rate: temperature < 30 degrees C, extracellular sodium < 70 mM, and membrane potential > +60 mV. EGTA concentration had no effect on outward INaCa with 100 mM cytoplasmic Na+ and 0.8 microM cytoplasmic Ca2+, conditions under which the current inactivated by about 70%. EGTA (0.1-10 mM) and BAPTA (10 mM) inhibited the current by about 80% when the outward INaCa was stimulated by 2 mM cytoplasmic ATP or by phosphatidylserine. The apparent Ki for EGTA was 0.2 mM. The electroneutral calcium ionophore, A23187, activated outward INaCa even in presence of 10 mM EGTA. Our results are consistent with EGTA acting as a simple calcium buffer with no direct effect on the exchanger. At low concentrations of EGTA, inhibition of the inward INaCa is expected due to submembrane calcium depletion by the exchanger; enhancement of the outward INaCa at low EGTA concentrations is expected because submembrane calcium accumulates and activates INaCa via regulatory calcium binding sites.

Adenosine Triphosphate↗
PubMed1995

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

Animals↗
PubMed1994

Inactivation of outward Na(+)-Ca2+ exchange current in guinea-pig ventricular myocytes.

1. Outward Na(+)-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membranes patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 microM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 +/- 2.6 s; n = 6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 microM sodium tetradecylsulphate, both of which decrease Na(+)-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 +/- 3.5 and 27.3 +/- 16.3 s, means +/- S.D., n = 11). Intracellular application of 0.5-2 mg ml-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches.

Ion transporters can be thought of as ion channels that open and close only at one end at a time. As in real channels, ions may cross through an electrical field as they diffuse into and bind within the transporter pore, thereby generating electrical current. Extracellular sodium binding by the sodium potassium (Na,K) pump is associated with ultrafast charge movements in giant cardiac membrane patches. The charge movements are complete within 4 microseconds. They occur only when binding sites are open to the extracellular side, and they are abolished by ouabain and by the removal of extracellular sodium. Fast extracellular ion binding may be the exclusive source of Na,K pump electrogenicity.

Adenosine Triphosphate↗
PubMed1993

Initial localization of regulatory regions of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

We have analyzed the regulatory properties of the wild-type cardiac Na(+)-Ca2+ exchanger expressed in Xenopus laevis oocytes using the giant excised patch technique. The exchanger is activated by cytoplasmic application of chymotrypsin and exhibits a number of properties that can be changed or abolished by chymotrypsin treatment, including cytoplasmic Na(+)-dependent inactivation, secondary regulation by free cytoplasmic Ca2+, and inhibition by exchanger inhibitory peptide. Thus, the cloned exchanger expressed in oocytes exhibits regulatory properties similar to those of the native sarcolemmal exchanger. The exchanger protein contains a large (520 amino acids) hydrophilic domain modeled to be intracellular. The role of this region in exchanger function and regulation was examined by deletion mutagenesis. Mutants with residues 240-679 and 562-685 deleted exhibited exchange activity, indicating that this extensive region is not essential for transport function. Both mutants were stimulated by chymotrypsin treatment. Neither mutant demonstrated regulation by free cytoplasmic Ca2+ (Ca2+i) or inhibition by exchanger inhibitory peptide (XIP). However, mutant delta 562-685 but not delta 240-679 displayed Na(+)-dependent inactivation. The data suggest that the binding sites for XIP and regulatory Ca2+ may reside in the region encompassed by residues 562-685. A chimera made from renal and cardiac exchangers has normal regulatory characteristics and helps to further define these sites.

Animals↗
PubMed1993

A novel method for direct application of phospholipids to giant excised membrane patches in the study of sodium-calcium exchange and sodium channel currents.

Effects of membrane phospholipids on Na(+)-Ca2+ exchange and Na+ channel currents were studied in giant excised cardiac sarcolemmal patches. Phospholipids were suspended in an inert vehicle of alpha-tocopherol acetate and hexane and were then directly applied to the side of patch electrodes at a short distance from the tip during current recording. Phosphatidylserine strongly stimulated outward Na(+)-Ca2+ exchange current and altered the kinetics of cytoplasmic Na(+)- and Ca(2+)-dependent secondary modulation. This effect was partially reversed by phosphatidylcholine. Prolonged treatment with phosphatidylserine eliminated the inactivation transient normally observed upon rapid application of cytoplasmic Na+ but left cytoplasmic Ca2+ dependence largely intact. In such cases, subsequent chymotrypsin treatment removed cytoplasmic Ca2+ dependence, but had no further stimulatory effect, indicating maximum alleviation of inactivation by phosphatidylserine. While these results indicate that phosphatidylserine acts on a cytoplasmic, protease-sensitive regulatory domain of the exchanger, phosphatidylserine also stimulated the exchange current after deregulation by chymotrypsin, indicating an effect on the exchange mechanism itself. As in other myocyte preparations, cardiac Na+ currents in giant patches undergo a time-dependent negative shift in the voltage dependence of steady-state inactivation. Loss of phosphatidylserine from the cytoplasmic leaflet (i.e. loss of transbilayer asymmetry of phosphatidylserine distribution) does not appear to be the underlying cause, since phosphatidylserine did not reverse this shift, despite stimulation of Na(+)-Ca2+ exchange current in the same patches.

Animals↗
PubMed1992

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation.

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).

Adenosine Triphosphate↗
PubMed1992

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Secondary modulation by cytoplasmic calcium and ATP.

Dynamic responses of cardiac sodium-calcium exchange current to changes of cytoplasmic calcium and MgATP were monitored and analyzed in giant membrane patches excised from guinea pig myocytes. Secondary dependencies of exchange current on cytoplasmic calcium are accounted for in terms of two mechanisms: (a) The sodium-dependent inactivation process, termed I1 modulation, is itself strongly modulated by cytoplasmic calcium. Recovery from the I1 inactivated state is accelerated by increasing cytoplasmic calcium, and the calculated rate of entrance into I1 inactivation is slowed. (b) A second modulation process, termed I2 modulation, is not sodium dependent. As with I1 modulation, the entrance into I2 inactivation takes place over seconds in the absence of cytoplasmic calcium. The recovery from I2 inactivation is a calcium-dependent transition and is rapid (< 200 ms) in the presence of micromolar free calcium. I1 and I2 modulation can be treated as linear, independent processes to account for most exchange modulation patterns observed: (a) When cytoplasmic calcium is increased or decreased in the presence of high cytoplasmic sodium, outward exchange current turns on or off, respectively, on a time scale of multiple seconds. (b) When sodium is applied in the absence of cytoplasmic calcium, no outward current is activated. However, the full outward current is activated within solution switch time when cytoplasmic calcium is applied together with sodium. (c) The calcium dependence of peak outward current attained upon application of cytoplasmic sodium is shifted by approximately 1 log unit to lower concentrations from the calcium dependence of steady-state exchange current. (d) The time course of outward current decay upon decreasing cytoplasmic calcium becomes more rapid as calcium is reduced into the submicromolar range. (e) Under nearly all conditions, the time courses of current decay during application of cytoplasmic sodium and/or removal of cytoplasmic calcium are well fit by single exponentials. Both of the modulation processes are evidently affected by MgATP. Similar to the effects of cytoplasmic calcium, MgATP slows the entrance into I1 inactivation and accelerates the recovery from inactivation. MgATP additionally slows the decay of outward exchange current upon removal of cytoplasmic calcium by 2-10-fold, indicative of an effect on I2 inactivation. Finally, the effects of cytoplasmic calcium on sodium-calcium exchange current are reconstructed in simulations of the I1 and I2 modulation processes as independent reactions.

Adenosine Triphosphate↗
PubMed1992

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Ion and voltage dependencies of the transport cycle.

Ion and voltage dependencies of sodium-calcium exchange current were studied in giant membrane patches from guinea pig ventricular cells after deregulation of the exchanger with chymotrypsin. (a) Under zero-trans conditions, the half-maximum concentration (Kh) of cytoplasmic calcium (Cai) for activation of the isolated inward exchange current decreased as the extracellular sodium (Nao) concentration was decreased. The Kh of cytoplasmic sodium (Nai) for activation of the isolated outward exchange current decreased as the extracellular calcium (Cao) concentration was decreased. (b) The current-voltage (I-V) relation of the outward exchange current with saturating concentrations of Nai and Cao had a shallow slope (twofold change in approximately 100 mV) and a slight saturation tendency at very positive potentials. The outward current gained in steepness as the Nai concentration was decreased, such that the Kh for Nai decreased with depolarization. The decrease of Kh for Nai with depolarization was well described by a Boltzmann equation (e alpha.Em/26.6) with a slope (alpha) of -0.06. (c) Voltage dependence of the outward current was lost as the Cao concentration was decreased, and the Kh for Cao increased upon depolarization with a Boltzmann slope of 0.26. (d) The I-V relation of the inward exchange current, under zero-trans conditions, was also almost linear (twofold change in approximately 100 mV) and showed some saturation tendency with hyperpolarization as the Cai concentration was decreased. The Kh for Cai decreased with depolarization (Boltzmann slope, -0.10). Voltage dependence of the inward current was decreased in the presence of a high (300 mM) Nao concentration. (e) In the presence of both Na and Ca on both membrane sides, the I-V relations with saturating Nai show sigmoidal shape and clear saturation at positive potentials. Measured reversal potentials were close to the equilibrium potential expected for a 3 Na to 1 Ca exchange. (f) Nai and Cai interacted competitively with respect to the outward current, but in a mixed competitive-noncompetitive fashion with respect to the inward current. (g) Cai inhibited the outward exchange current in a voltage-dependent manner. The half-effective concentration for inhibition (Ki) by Cai increased upon depolarization with a Boltzmann slope of 0.32 in 25 mM Nai and 0.20 in 100 mM Nai. (h) Nai also inhibited the inward exchange current voltage dependently. The Ki decreased upon depolarization (Boltzmann slope, -0.11 at 3 microM Cai and -0.10 at 1.08 mM Cai).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

The giant cardiac membrane patch method: stimulation of outward Na(+)-Ca2+ exchange current by MgATP.

1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na(+)-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 G omega seals with 14-24 microns pipette tip diameters) excised from guinea-pig, rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na(+)-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 microns), (iii) fast, rigorous concentration control and (iv) Na(+)-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na(+)-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 microns-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mM or greater. 8. Stimulation of Na(+)-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 microM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

Mechanism of cardiac Na(+)-Ca2+ exchange current stimulation by MgATP: possible involvement of aminophospholipid translocase.

1. The sensitivity of outward Na(+)-Ca2+ exchange current to charged amphiphiles and phospholipids was tested in giant excised inside-out membrane patches from guinea-pig and rabbit myocytes. 2. Screening of membrane surface potentials with dimethonium (10 mM), spermine (200 microM) and spermidine (100 microM) was without effect, while the positively charged ionic detergents hexadecyltrimethylammonium and dodecyltrimethylammonium strongly inhibited steady-state outward exchange current (0.1-10 microM). 3. Interventions expected to increase negative surface charge included treatment of the cytoplasmic surface with phospholipase D, application of dodecylsulphate (1-10 microM), application of the short-chain phosphatidylserine derivative, dicapryl phosphatidylserine (C10PS), and inclusion of 1-3% phosphatidylserine in the hydrocarbon mixture used to coat electrodes. Each intervention strongly stimulated Na(+)-Ca2+ exchange current in a similar way to MgATP, reducing the fractional decay of outward exchange current (inactivation) during application of high cytoplasmic sodium. 4. The MgATP-stimulated exchange current was inhibited with a Ki of approximately 1 microM by pentalysine, which is known to associate with phosphatidylserine head groups. After 'deregulation' of the exchanger by chymotrypsin, pentalysine was without effect. 5. Inclusion in the pipette of 0.2 mM-pyridyldithioethylamine (an oxidizing inhibitor of aminophospholipid translocase) abolished stimulation of outward exchange current by MgATP without inhibiting basal outward exchange current or sodium pump current. 6. Application to the cytoplasmic side of 1.5 mM-diamide, which reportedly decreases membrane phospholipid asymmetry, apparently reversed the effect of MgATP. After treatment with diamide and subsequently with dithiothreitol, Na(+)-Ca2+ exchange current was again stimulated by MgATP. Diamide was without effect when secondary exchange regulation had been previously removed by chymotrypsin. 7. Potassium current carried by the surface potential-sensitive ionophore, nonactin, was stimulated by MgATP when extracellular surface charge had been neutralized. The effect was largest (40-90%) when low ionic strength cytoplasmic solutions were employed, consistent with an increase of negative membrane charge on the cytoplasmic side during MgATP application. 8. Potassium current carried by nonactin was inhibited by MgATP when cytoplasmic surface charge had been neutralized and extracellular solutions of low ionic strength were employed, consistent with a decrease of negative membrane charge on the extracellular side. 9. These results indicate that the stimulatory effect of MgATP on Na(+)-Ca2+ exchange current could involve changes of charged membrane lipids, that the effect probably involves a transmembrane, oxidation-sensitive protein, that pentalysine-sensitive sites are involved, that phosphatidylserine mimics the effect of MgATP, and that the effect extends to a simple surface potential-sensitive ionophore.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger.

Na+/Ca2+ exchange is electrogenic and moves one net positive charge per cycle. Although the cardiac exchanger has a three-to-one Na+/Ca2+ stoichiometry, details of the reaction cycle are not well defined. Here we associate Na+ translocation by the cardiac exchanger with positive charge movement in giant membrane patches from cardiac myocytes and oocytes expressing the cloned cardiac Na+/Ca2+ exchanger. The charge movements are initiated by step increments of the cytoplasmic Na+ concentration in the absence of Ca2+. Giant patches from control oocytes lack both steady-state Na+/Ca2+ exchange current (INaCa) and Na(+)-induced charge movements. Charge movements indicate about 400 exchangers per micron 2 in guinea-pig sarcolemma. Fully activated INaCa densities (20-30 microA cm-2) indicate maximum turnover rates of 5,000 s-1. As has been predicted for consecutive exchange models, the apparent ion affinities of steady state INaCa increase as the counterion concentrations are decreased. Consistent with an electroneutral Ca2+ translocation, we find that voltage dependence of INaCa in both directions is lost as Ca2+ concentration is decreased. The principal electrogenic step seems to be at the extracellular end of the Na+ translocation pathway.

Animals↗
PubMed1991

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.

Amino Acid Sequence↗
PubMed1990

Regulation and deregulation of cardiac Na(+)-Ca2+ exchange in giant excised sarcolemmal membrane patches.

A plasmalemmal Na(+)-Ca2+ exchange mechanism is an important electrogenic determinant of contractility in cardiac cells. As in other cell types, calcium influx by Na(+)-Ca2+ exchange is secondarily activated by cytoplasmic calcium and probably ATP, but these modulatory mechanisms are either absent or altered in isolated cardiac sarcolemmal vesicles. Involvement of a calcium-dependent protein kinase in exchange regulation has been suggested but not verified. Here I describe measurements of outward Na(+)-Ca2+ exchange current, corresponding to calcium influx, in giant excised sarcolemmal patches from guinea pig myocytes. The exchange current is stimulated by both calcium and Mg-ATP from the cytoplasmic face, evidently through separate mechanisms. Activation by cytoplasmic calcium takes place within seconds, is reversible, and does not require ATP. Stimulation by Mg-ATP reverses only slowly over greater than 10 min, or not at all. Unexpectedly, a substantial decrease in exchange current occurs during activation by cytoplasmic sodium, which seems to reflect an inactivation process rather than ion concentration changes or a 'first pass' exchange cycle. This apparent inactivation, and the modulations by cytoplasmic calcium and Mg-ATP, are all abolished by brief treatment of the cytoplasmic surface with chymotrypsin, leaving the exchanger in a maintained state of high activity. Therefore, limited proteolysis deregulates Na(+)-Ca2+ exchange and could contribute to the loss of secondary regulation of the exchange in isolated sarcolemmal vesicles.

Adenosine Triphosphate↗
PubMed1989

Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents.

A tightly-sealed cardiac sarcolemmal patch preparation of large diameter (10-16 microns with 5-25 G delta seals) has been developed to study regulation of selected electrogenic mechanisms. Formation of a large readily accessible membrane surface area is achieved by incubating freshly isolated myocytes in a high KCl/zero calcium solution, which promotes separation of sarcolemma from myofilaments. The formation of large-diameter, high resistance seals is facilitated by depositing a neutral hydrocarbon film on electrode tips. Sodium currents in excised patches are stable for patch life-times of 15-40 min with peak current densities of 120 to 350 microA/cm2. Outward sodium-calcium exchange current is identified by its specific dependence on external calcium and internal sodium, inhibition by cobalt and dichlorobenzamil, and activation by less than 0.1 microM internal free calcium. Maximum exchange current density is greater than 20 microA/cm2.

Amiloride↗
PubMed1989

Slowly relaxing caffeine responses in rat ventricle: relationships of ryanodine and caffeine actions.

Contractile responses to 20 mM caffeine were compared in arterially perfused rat right ventricle as tension, in intact rat myocytes as shortening, and in Triton X-100-"skinned" rat myocytes as shortening and stiffness. Responses in intact quiescent ventricle and membrane-disrupted myocytes at -log molar Ca concentration (pCa) of 7.0-6.8 were similar; force and stiffness rose quickly on application of caffeine and declined incompletely toward base line over 20 min. Subsequent caffeine responses were blunted. By contrast, in freshly isolated rat myocytes, rapid caffeine exposures induced large phasic contractures, which relaxed for the most part in 2-5 s. These responses were largely suppressed by 0.2 microM ryanodine pretreatment at 35 degrees C but not by high ryanodine concentrations (10 microM). Ryanodine was without effect on slowly relaxing contractile responses in both intact ventricle and Triton X-100-treated myocytes. The results are discussed in relation to the predicted effects of a calcium store leak in a simple model of cardiac excitation-contraction coupling. The results suggest that slowly relaxing caffeine contractures can be caused entirely by direct actions of caffeine on myofilaments.

Alkaloids↗