Biomedical subjects
Publications and source records attributed to G Isenberg.
Analyses of dynamic light scattering data by stretched exponential fit show that vinculin has a negligible influence on internal actin filament dynamics and actin bending stiffness which contrasts with our previous observations with talin, another actin and vinculin-binding protein from focal adhesions. The results here agree with kinetic and rheologic measurements.
1. Myocytes from the urinary bladder were stretched longitudinally by 5-20%. At -50 mV, stretch induced whole-cell inward currents (Iin) between -100 and -600 pA. Iin decayed slowly with time to 93 +/- 20% (mean +/- S.E.M., n = 6) of the initial value in 1 min. The mechanisms of this 'adaptation' and its modulation by dibutyryl cAMP (dBcAMP) were analysed with whole-cell and single channel currents. 2. When the cells were internally perfused with 100 microM 8-bromo-cAMP (8BrcAMP), stretch induced an Iin of the usual amplitude that decayed completely within 40 +/- 13 s. When 200 microM dBcAMP was bath applied 10 s after the start of the stretch, Iin decayed to zero within 85 +/- 18 s. 3. dBcAMP increased the K+ current through Ca(2+)-activated BK channels (IK(Ca)) at 0 mV with a time course that correlated well with the decay of Iin, and block of IK(Ca) by TEA suppressed the dBcAMP-induced decay of Iin. In the presence of intracellular BAPTA, dBcAMP increased the stretch-induced Iin. The results suggest that adaptation is caused by superimposition of IK(Ca) which is increased through elevation of near-membrane [Ca2+] and by cAMP-dependent phosphorylation. 4. Single channel analysis was carried out with 140 mM KCl electrode solution and at -50 mV. Stretch-activated channels (SACs) were recorded during pulses of negative pressures between -2 and -5 kPa. Activity (NPo) of SACs was constant for at least 4 min, e.g. evidence for adaptation was missing. dBcAMP (200 microM) increased NPo of SACs by 142 +/- 35% (n = 16). 5. dBcAMP increased NPo via frequency of openings and channel open time. In five of sixteen patches, dBcAMP induced openings without suction. Similar effects were induced by the catalytic subunit of cAMP-dependent protein kinase (PKAc), applied to inside-out patches. 6. NPo, normalized by its maximum, increased with more negative pressure along an S-shaped curve. dBcAMP increased the sensitivity of SACs to stretch by shifting the point of half-maximal activity from -3.2 to -2.6 kPa. 7. The augmentation of NPo by dBcAMP is attributed to the phosphorylation of SACs promoting their opening. Adaptation of Iin is discussed as a 'secondary' effect of stretch-activated channels: Ca2+ influx through SACs increases the Ca2+ concentration that activates BK channels whose Ca2+ sensitivity is increased by cAMP.
In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.
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1. In guinea-pig ventricular cells, the concentration of ionized cytosolic calcium ([Ca2+]o) was estimated from the fluorescence of 100 microM K5-indo-1. At 36 degrees C and 2 mM [Ca2+]o, the Ca2+ load of the cells was varied by 1 Hz trains of conditioning clamp pulses to -30 mV (low Ca2+ load), 0 mV (intermediate Ca2+ load) and paired pulses (high Ca2+ load). After seven pulses potentiation was steady and short test pulses to 0 mV were tested for their efficacy in triggering [Ca2+]c transients. The influx of trigger Ca2+ was graded by varying the test-pulse duration between 1 and 180 ms. 2. After a 3 min rest period, [Ca2+]c was 100 +/- 20 nM (mean +/- S.E.M.) and 2 ms test pulses were unable to induce [Ca2+]c transients. Test pulses of 2 ms duration, however, induced [Ca2+]c transients after potentiation with single or paired pulses. 3. At high cellular Ca2+ load, the amplitude of the [Ca2+]c transients (delta[Ca2+]c) gradually increased with pulse durations up to 8 ms. Pulse durations between 8 and 160 ms, however, did not further increase delta[Ca2+]c as if the largest part of the [Ca2+]c transient was due to regenerative contribution of Ca(2+)-induced Ca2+ release. 4. Pulses of 160 ms duration induced 'saturating' responses whose amplitudes delta[Ca2+]c, t = infinity decreased from 938 +/- 120 nM at high Ca2+ load, to 610 +/- 90 and 350 +/- 120 nM at intermediate and low Ca2+ loads, respectively. 5. Delta[Ca2+]c was more sensitive to the duration of Ca2+ influx at low or intermediate Ca2+ loads than at high Ca2+ load. When delta[Ca2+]c was plotted against the test-pulse duration, 50% of delta[Ca2+]c, t = infinity was found to be at 9 +/- 2 ms (low), 4.6 +/- 1 ms (intermediate) or 1.8 +/- 0.5 ms pulses (high Ca2+ load). Correspondingly, the efficacy of 2 ms test pulses in triggering [Ca2+]c transients increased with the Ca2+ load. 6. At high Ca2+ load, [Ca2+]c peaked nearly independently of pulse duration at 19 +/- 3 ms. At intermediate or low Ca2+ load, time to peak increased with pulse duration. 7. The results confirm the theory that sarcoplasmic reticulum (SR) Ca2+ release contributes an amount to the [Ca2+]c transient that increases with the cellular Ca2+ load. The results are compatible with the hypothesis that SR Ca2+ release can be activated by both Ca2+ influx and by SR Ca2+ release and that the latter mechanism constitutes a positive feedback, the amplification of which increases with the amount of releasable Ca2+.
1. This study tests the hypothesis that whole-cell cardiac SR Ca2+ release is graded by recruitment of independent 'release units'. Structurally, an individual release unit may comprise ca four sarcolemmal L-type Ca2+ channels, adjacent ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels and the junctional gap between them. After depolarization, the first opening of a single L-type Ca2+ channel of the unit provides sufficient Ca2+ influx to increase local [Ca2+] beyond the threshold activating Ca(2+)-induced Ca2+ release (CICR), which amplifies local [Ca2+] until all release channels of the unit are active. This all-or-none activation does not spread to other release units. Gradation of whole-cell Ca2+ release is predicted to correlate with the cumulative probability density distribution of first latency of L-type Ca2+ channels or the activation time course of the calcium current, ICa. 2. Guinea-pig ventricular myocytes were potentiated by paired voltage-clamp pulses (1 Hz, 2 mM [Ca2+]o, 40 microM K5-indo-1, 36 degrees C). When the cellular Ca2+ load was at a steady high level, cytosolic calcium concentration ([Ca2+]c) transients were measured in response to test pulses of varied pulse duration (PD, 1-180 ms) and amplitude (-20, 0, 20 and 50 mV). The maximal rate of rise (RRmax) of the [Ca2+]c transient was used as an indicator for SR Ca2+ release. 3. Fast [Ca2+]c transients due to 4 ms pulses to 0 or 50 mV were blocked by 1 mM cadmium suggesting that these Ca2+ release signals are triggered by Ca2+ influx through L-type Ca2+ channels and not by Ca2+ influx through Na(+)-Ca2+ exchange. 4. RRmax increased with longer PD along the sigmoidal curve [1-exp(-PD/tau)]kappa(exponent k: 2 < k < 3). The time constant, tau, resembled the activation time constant of whole-cell ICa (Cs(+)-dialysed cells). A PD longer than a limiting duration did not modify RRmax. That is, inactivation of ICa was not reflected in the duration dependence. 5. Single L-type Ca2+ channels (cell-attached patches, 36 degrees C, -20 mV, 3.6 mM CaCl2 and 1 microM Bay K 8644 in patch pipette) opened with a waiting time the cumulative probability distribution of which resembled the duration dependence of RRmax, suggesting that the first opening of L-type Ca2+ channels determines whether the corresponding release unit contributes to the [Ca2+]c transient activated during a short voltage-clamp pulse. 6. The time constant, tau, of the duration dependence was shorter at positive than at negative potentials.(ABSTRACT TRUNCATED AT 400 WORDS)
1. By means of two patch-pipettes, isolated urinary bladder myocytes were longitudinally stretched up to 20% beyond slack length (delta L = 20%). 2. Experiments were conducted using both voltage and current clamp configurations. In current clamped cells at 23 degrees C, delta L depolarized the membrane from -50 to ca -15 mV, the amplitude of depolarization increasing with the extent of delta L. At 36 degrees C, delta L induced action potentials or increased the frequency of spontaneous action potentials. 3. In voltage clamped cells at a holding potential of -50 mV, stretch induced an inward current (Iin) and increased the input conductance. Both effects increased with delta L. They were blocked by 40 microM gadolinium, suggesting stretch activation of non-selective cation channels (SACs) as the underlying mechanism. 4. Stretch-induced difference currents rectified outwardly and reversed at a reversal potential (Erev) of -28 +/- 10 mV. Twenty millimolar [TEA]o suppressed the rectification and shifted Erev to 0 +/- 1 mV. The result suggests that stretch can activate not only SACs but also TEA-sensitive K+ channels. 5. Stretch changed the net current due to clamp steps to 0 mV as though it increased the potassium current (IK) and reduced the calcium current (ICa). While 20 mM intracellular BAPTA did not modify the stretch-induced whole-cell inward current (Iin) at -50 mV, it suppressed the stretch effects on IK and ICa as if these effects were mediated by an increase in the subsarcolemmal Ca2+ concentration. 6. The results support the hypothesis that longitudinal stretch can activate SACs and Ca2+ influx through them. In non-clamped cells, stretch can also modulate Ca2+ influx through L-type Ca2+ channels via changes in membrane potential.
The interaction of the actin-binding protein filamin with mixtures of zwitterionic and anionic phospholipids (DMPC, DMPG, PC, PS) was studied in reconstituted lipid monolayers and bilayers. Protein-lipid interactions were investigated by differential scanning calorimetry, the film balance technique, and hydrophobic photoradiolabeling. For calorimetric assays, multilamellar vesicles (MLVs) and large unilamellar vesicles produced by the extrusion technique (LUVETs) were used. With MLVs, filamin induced a pronounced drop in phase transition cooperativity. Mixed DMPC/DMPG LUVETs showed a linear decrease of the main phase transition enthalpy and a significant shift in temperature for the solidus and liquidus lines with increasing mole fractions of reconstituted filamin. The insertion of native filamin into uncharged and negatively charged lipid monolayers was measured in time/area diagrams with the film balance technique. Finally, we have newly synthesized a highly sensitive lipid analogue, [125]TID-PC/16, which selectively labels membrane-embedded hydrophobic domains of proteins, and which proved to label filamin, supporting evidence that this protein partially inserts into the hydrophobic domain of liposomes.
The cytoskeletal protein talin potentially plays a key role in actin-membrane linkage. It is able to nucleate actin filament growth in vitro while binding simultaneously to lipid bilayers. Thrombin digestion of human platelet talin yields tow polypeptide domains of 200 kDa and 47 kDa. We have purified these fragments and analyzed their functional properties: the 200-kDa fragment was active in nucleating actin filament formation and reduced the viscosity of filamentous actin, comparable to the effects of the intact protein. The 47-kDa fragment was inactive in this respect. However, the 47-kDa polypeptide, but not the 200-kDa fragment, interacted specifically with large liposomes containing acidic phospholipids. This is demonstrated by selective, hydrophobic photolabeling of the 47-kDa fragment using phosphatidylserine liposomes containing trace amounts of a photoactivable phospholipid analogue and by selective co-sedimentation of this domain with the liposomes. The 200-kDa fragment, whether alone or in conjunction with the small fragment, neither incorporated significant amounts of label nor co-sedimented with the liposomes. We thus are able to attribute specialized functions to distinct domains on the talin molecule. These enable the protein to interact simultaneously with actin filaments and lipid membranes.
The reaction of smooth muscle filamin and skeletal muscle actin was kinetically examined by double exponential analysis. The overall rate of binding, k+1, is concentration and temperature dependent whilst the overall rate of cross-linking/bundling, k+2, is concentration independent. The activation energy, Ea = 99.5 kJ/mol, was calculated from the Arrhenius Plot.
It is concluded from structure predictions of the primary amino acid sequence by computer analyses that two segments of non-muscle filamin could facilitate lipid membrane attachment or anchoring. Residues 49-71 of the amino-terminal may attach to phospholipid membranes, and residues 131-155 may anchor in the hydrophobic region of lipid membranes.
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L-type Ca2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intracellular pH (pHi). Whole cell ICa fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36 degrees C), and increased when pHi was made more alkaline by addition of 20 mM NH4Cl. Peak ICa was less pHi sensitive than late ICa (170 ms after depolarization to 0 mV). pHi-effects on single Ca2+ channel currents were studied with 110 mM BaCl2 as the charge carrier (22 degrees C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4Cl or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re-openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca2+ homeostases and vasotonus is discussed.
Modulation of L-type Ca2+ channel current by extracellular pH (pHo) was studied in vascular smooth muscle cells from bovine pial and porcine coronary arteries. Relative to pH 7.4, alkaline pH reversibly increased and acidic pH reduced ICa. The efficacy of pHo in modulating ICa was reduced when the concentration of the charge carrier was elevated ([Ca2+]o or [Ba2+]o varied between 2 and 110 mM). Analysis of whole cell and single Ca2+ channel currents suggested that more acidic pHo values shift the voltage-dependent gating (approximately 15 mV per pH-unit) and reduce the single Ca2+ channel conductance gCa due to screening of negative surface charges. pHo effects on gCa depended on the pipette [Ba2+] ([Ba2+]p), pK*, the pH providing 50% of saturating conductance, increased with [Ba2+]p according to pK* = 2.7-2.log ([Ba2+]p) suggesting that protons and Ba2+ ions complete for a binding site that modulates gCa. The above mechanisms are discussed in respect to their importance for Ca2+ influx and vasotonus.
Contractile tension in response to 200-ms voltage-clamp pulses was measured in isolated guinea pig ventricular cells conditioned to constant Ca load. At 23 degrees C, the tension-voltage relation was bell shaped, decaying from a maximum at +20 mV to zero at +100 mV, but at 35 degrees C it was sigmoidal, with similar twitch tensions at +20 and +100 mV. Tension at 35 degrees C and +100 mV was reduced by ryanodine or caffeine and abolished by removal of Ca just before the test pulse. At 35 degrees C and +100 mV, twitch tension increased markedly as the Na concentration in the patch pipette ([Na]p) was varied between 0 and 20 mM. Cd (300 microM) blocked tension at all potentials at 23 degrees C, but tension remained in the presence of Cd at 35 degrees C (29% of control at +2 mV and 100% of control at +100 mV). Cd-resistant tension began to relax during the clamp pulse at all potentials (80 +/- 10 ms at +2 mV and 140 +/- 12 ms at +100 mV). Ni (3.6 mM) both reduced and slowed tension transients at all potentials. The results suggest that fast contractions due to sarcoplasmic reticulum Ca release can be triggered by Ca influx through either Ca current (ICa) or Na/Ca exchange and that those triggered through exchange are much more temperature sensitive than those triggered by ICa.
We ascertained by the stopped flow method the overall association rate constant, k+1, of filamin and alpha-actinin to fluorescently labelled filamentous actin of approximately 1.3 x 10(6) M-1.s-1 and approximately 1.0 x 10(6) M-1.s-1 as well as the overall dissociation rate constant, k-1, of approximately 0.6 s-1 and approximately 0.4 s-1, respectively. The overall equilibrium constant, K, for filamin and alpha-actinin to actin deduced from the relation K = k+1/k-1 agree well with published data.
Fluorescently labelled smooth muscle talin like native talin interacts with negatively or partly negatively charged lipid monolayers. This was measured in time/area diagrams using the film balance technique combined with fluorescence imaging after double photolabelling of talin and phospholipids.
In cardiac and smooth muscle cells, nonselective cation channels can be activated by hormones and neurotransmitters, by cell stretch, and by changes in membrane potential. Activation of nonselective cation channels can depolarize the cell membrane, induce Ca2+ influx through voltage-gated Ca2+ channels and contraction. Activation of nonselective cation channels may trigger contraction even when membrane depolarization is absent or when voltage-gated Ca2+ channels are blocked, provided the Ca2+ permeability of these channels is sufficiently high.