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  • 1
    Electronic Resource
    Electronic Resource
    Whole-Cell Membrane Currents from Human Osteoblast-like Cells (1998)
    Springer
    Calcified tissue international 62 (1998), S. 122-132 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Patch clamp electrophysiology — MG63 — Bone cell — Potassium channel.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Bone cells share common responses to external stimuli with most other cells. Among these are changes in membrane ion channel activity, although at present, relatively little is known about their nature or significance in human bone cells. Using the whole-cell configuration of the patch-clamp technique, we have revealed two types of membrane current in MG63 human osteoblast-like cells. With a potassium-based dialysis solution and a holding potential of −40 mV, voltage commands to more negative potentials elicited an inward current. This current showed little inactivation with time during the command pulse and exhibited some characteristics of an inwardly rectifying K current, including sensitivity to external K and Ba. The second type of current was outward, activated by depolarizing pulses from −40 mV. This current was transient in nature, activating in the first 50 ms of the pulse and then showing rapid inactivation to reach a steady-state level after 4 to 5 seconds. The transient outward current was sensitive to block by TEA, CTX, and to a lesser extent, Ba. These data suggest that a large proportion of this outward current is carried by K ions through channels that may be sensitive to the internal Ca ion concentration. The transient outward current was enhanced by setting the holding potential at −100 mV, and greatly inactivated by setting it at 0 mV. Increased understanding of the significance of these membrane currents may allow development and use of agents to modulate their action and therefore influence bone cell behavior in disease states such as osteoporosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900405
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  • 2
    Electronic Resource
    Electronic Resource
    Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 581-590 
    Details
    ISSN: 1432-2013
    Keywords: Key words Delayed rectifier ; FRCRCFa ; Inward rectifier ; L-type calcium current ; Myocyte ; Myristyl-FRCRCFa ; Na-Ca exchange ; Rabbit ventricle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, ”Myristyl- (Myr-) FRCRCFa”. Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35–37°C. The Na-Ca exchange current (I Na-Ca), L-type calcium current (I Ca,L), inward rectifier potassium current (I K1) and delayed rectifier potassium current (I K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, I Na-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of –40 mV, between +80 and –120 mV (ramp velocity 0.1 V s–1). In untreated cells, I Na-Ca at +60 mV was 7.1±0.6 pA/pF and at –100 mV was –2.7±0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 µM Myr-FRCRCFa, I Na-Ca was reduced to 4.2±0.3 pA/pF at +60 mV and –1.5±0.2 pA/pF at –100 mV (P〈0.02; n=7). After incubation with 20 µM Myr-FRCRCFa for 1 h, I Na-Ca at both potentials was further reduced (2.3±0.8 pA/pF at +60 mV; –0.9±0.3 pA/pF at –100 mV; P〈0.008 compared with control; n=4). Under selective recording conditions for I Ca,L, there was little difference in I Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I Ca,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of I Na-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an I Na-Ca blocker. I Ca,L, I K1 and I K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050675
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  • 3
    Electronic Resource
    Electronic Resource
    Characteristics of a transient outward current (sensitive to 4-aminopyridine) in Ca2+-tolerant myocytes isolated from the rabbit atrioventricular node (1999)
    Springer
    Pflügers Archiv 438 (1999), S. 68-78 
    Details
    ISSN: 1432-2013
    Keywords: Key words 4-AP ; Atrioventricular node ; Flecainide ; Ito ; K current ; Myocyte ; Quinidine ; Transient outward current
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  A transient outward current (I to) has been observed in the atrioventricular node (AVN), but its characteristics in Ca-tolerant AVN myocytes have not been investigated previously. In this study, I to was measured from Ca-tolerant rabbit AVN myocytes at 37°C, using the whole-cell patch-clamp technique. With interfering currents inhibited, 500-ms voltage-clamp pulses applied from –80 mV elicited I to at potentials positive to –30 mV, which increased in magnitude with test potential amplitude. This current was completely blocked by external application of 5 mM 4-aminopyridine (4-AP). During a command pulse, I to activated rapidly then inactivated with a bi-exponential time-course. Fast and slow time constants of current inactivation (τf and τs, respectively) showed voltage dependence. At 0 mV, τf was 14.5±2.7 ms and τs was 112.8±21.2 ms, whilst at +60 mV τf was 6.7±1.1 ms and τs was 63.7±9.2 ms (n=25). The steady-state inactivation relationship showed half-maximal inactivation at –33.8 mV (n=8). Re-activation of I to after an inactivating pre-pulse showed a bi-exponential time-course of recovery: τ1 was 196±70 ms, and τ2 was 2707±1010 ms (n=6, at –80 mV). Repetitive application of voltage-clamp test pulses showed that I to inactivation accumulated on repetitive stimulation, but reached a steady state rapidly for a given pulse frequency (0.2–1.0 Hz). AVN I to was sensitive to the class 1 anti-arrhythmic flecainide (EC50 for peak current of 24 µM), which showed selectivity for the rapidly inactivating current component. Quinidine also inhibited I to in a dose-dependent fashion, but did not affect the current time-course. Under voltage-clamp conditions, a simulated diastolic depolarisation from –70 to –45 mV did not significantly reduce I to amplitude, and under current-clamp conditions 4-AP inhibited spontaneous action potentials. Although this is consistent with a significant role for I to in shaping AVN activity, under the conditions of this study 4-AP also partially blocked the ”rapid” delayed rectifier current, I Kr, and so the effects of 4-AP on action potentials could not be attributed exclusively to its effects on I to.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050881
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  • 4
    Electronic Resource
    Electronic Resource
    An investigation of the role played by the E-4031-sensitive (rapid delayed rectifier) potassium current in isolated rabbit atrioventricular nodal and ventricular myocytes (1999)
    Springer
    Pflügers Archiv 438 (1999), S. 843-850 
    Details
    ISSN: 1432-2013
    Keywords: Action potential clamp Barium-sensitive current Cardiac repolarisation E-4031-sensitive current Inward rectifier potassium current Rapid delayed rectifier potassium current
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. The aim of this study was to measure and compare the profile of rapid delayed rectifier potassium current (I Kr) elicited by action potential (AP) waveforms applied to isolated rabbit atrioventricular nodal (AVN) and ventricular myocytes. All measurements were made using whole-cell patch-clamp recordings at 37°C. In AVN myocytes, I Kr during voltage steps and slow ramp depolarisations showed "inward rectification" (characteristic for this channel) at positive potentials. The E-4031-sensitive current showed half-maximal activation at –10.8±0.86 mV, with a slope factor for the activation relation of 6.5±0.77 mV (n=7). During AVN APs, I Kr rapidly reached a peak after the AP upstroke and remained at similar amplitude until late in AP repolarisation. At the maximum diastolic potential following the AVN AP, a component of I Kr remained which decayed during the pacemaker depolarisation, consistent with a role for the current in generating AVN pacemaker activity. In ventricular myocytes I Kr was small at the beginning of the AP, and increased slowly during the AP plateau. Measurement of Ba-sensitive-inward rectifier K current (I K1) in ventricular myocytes revealed that I K1 rapidly increased during the final AP repolarisation phase, whilst I Kr declined. It is concluded that I Kr may participate in both AP repolarisation and the pacemaker depolarisation in AVN cells, whilst in ventricular myocytes, I Kr and I K1 participate in controlling early and final AP repolarisation respectively.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004249900118
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  • 5
    Electronic Resource
    Electronic Resource
    Characteristics of the delayed rectifier K current compared in myocytes isolated from the atrioventricular node and ventricle of the rabbit heart (1996)
    Springer
    Pflügers Archiv 431 (1996), S. 713-722 
    Details
    ISSN: 1432-2013
    Keywords: Key words Atrioventricular node ; Myocyte ; Ventricle ; Delayed rectifier potassium current (Ik) ; E-4031
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The delayed rectifier potassium current (I K) is known to be important in action potential repolarisation and may contribute to the diastolic pacemaker depolarisation in pacemaker cells from the heart. In this study, using whole-cell patch clamp, we investigated the characteristics of I K in morphologically normal cells from the atrioventricular node (AVN) and ventricle of the rabbit heart. Cells were held at −40 mV and 5 μM external nifedipine was used to block L-type calcium current (I Ca,L). Significant I K was observed with pulses to potentials more positive than −30 mV. The steady-state activation curve in both cell types showed maximal activation at between + 10 and + 20 mV. Half-maximal activation of I K occurred at −4.9 and −4.1 mV with slope factors of 8.3 and 12.4 mV in ventricular and AVN cells, respectively. Using pulses of increasing duration, significant I K tails after repolarisation from + 40 mV were observed with pulses of 20 ms and increased with pulses up to 100–120 ms in both cell types. Pulses of longer duration did not activate further I K and this suggested that only the rapid component of I K, called I Kr, was present in either cell type. Moreover, I K tails after pulses to all potentials were blocked completely by E-4031, a selective blocker of I Kr. The reversal potential of I K varied with the concentration of external K. Superfusion of AVN cells with medium containing 4, 15 and 40 mM [K+]o resulted in reversal potentials of −81, −56 and −32 mV, respectively, which are close to values predicted if the I K channel were highly selective for K. The time constants for deactivation of I K in ventricle and AVN on return to −40 mV after a 500-ms activating pulse to + 60 mV were 480 ms and 230 ms, respectively. The faster deactivation of I K in AVN cells was a distinguishing feature and suggests that there may be differences in the I Kr channel protein between ventricular and AVN cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050056
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  • 6
    Electronic Resource
    Electronic Resource
    Time course and voltage dependence of expressed HERG current compared with native ”rapid” delayed rectifier K current during the cardiac ventricular action potential (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 843-853 
    Details
    ISSN: 1432-2013
    Keywords: Key words Action potential clamp ; Cardiac ; Delayed rectifier ; HERG ; Ikr ; Potassium channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  It is widely believed that HERG (human ether-a-go-go-related gene) encodes the major subunit of the cardiac ”rapid” delayed rectifier K channel. The aims of the present study were threefold: (1) to record directly the time course and voltage dependence of expressed HERG current in a mammalian cell line, during an imposed ventricular action potential (AP); (2) to compare this with native rapid delayed rectifier current (I Kr) elicited by applying an AP command to isolated guinea-pig ventricular myocytes; (3) to provide mechanistic information regarding the profile of HERG/I Kr during the AP. We used the AP clamp technique and conventional whole-cell patch-clamp recordings at 32–34°C. HERG was transiently expressed in Chinese hamster ovary (CHO) cells. There was an outward current in transfected CHO cells, which developed progressively during the AP plateau and slow repolarisation phase. The instantaneous current-voltage (I-V) relation for both leak-subtracted HERG current (n=10) and E-4031-sensitive current (n=6) during AP repolarisation was maximal between –30 mV and –40 mV. The conductance-voltage (G-V) relation was maximal at potentials between –60 and –75 mV. A similar voltage dependence for HERG current was observed during a descending ramp from +60 to –80 mV (n=5), but not during either an ascending ramp (n=5), or a reversed AP waveform (n=8). These data suggest that instantaneous HERG current during the AP does not depend on the instantaneous command voltage alone, but upon the previous voltages during the applied waveform. The time course of activation of HERG current at potentials near the AP plateau was rapid. Tail currents recorded on premature repolarisation at different time points in the AP showed directly that HERG also activates rapidly during the AP. The I-V profiles of fully activated HERG and of current during the AP were very similar. I Kr from guinea-pig ventricular myocytes was measured as E-4031-sensitive current during the AP clamp command. The current had a similar I-V and G-V profile to HERG current in CHO cells. These data indicate that HERG current and native I Kr are similar during an applied AP waveform. Activation of HERG is rapid during the AP. However, due to rapid inactivation relatively little current flows until the potential becomes less positive than 0 mV. The removal of inactivation then allows more current to flow, giving rise to the distinct instantaneous I-V profile during the AP. The correlation between the voltage dependence of HERG during the AP and the fully activated I-V relation indicates that the contribution of HERG/I Kr to AP repolarisation is more significantly determined by the open-channel I-V relation, than the precise activation time course of the current.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050713
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  • 7
    Electronic Resource
    Electronic Resource
    The Fura-2 transient can show two types of voltage dependence at 36°C in ventricular myocytes isolated from the rat heart (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 215-224 
    Details
    ISSN: 1432-2013
    Keywords: Key words Excitation-contraction coupling ; Na-Ca exchange ; Calcium transient ; cardiac myocyte
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We used the whole-cell patch-clamp method to investigate the voltage dependence of the L-type Ca current (I Ca,L) and intracellular Ca (Cai) transient in ventricular myocytes isolated from the rat heart. Intracellular Ca was monitored using Fura-2 and the experiments were carried out at 36° C. We measured I Ca,L by using a caesium-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak I Ca,L amplitude was bell-shaped: I Ca,L was maximal at +10 mV and declined at more positive potentials. When I Ca,L was integrated over the first 25 ms to estimate the magnitude of Ca entry, this had a very similar voltage dependence to peak I Ca,L. In all cells, phasic Fura-2 transients were abolished by 5 μM ryanodine (a blocker of the sarcoplasmic reticulum, SR) showing that the Fura-2 transient provided an index of the magnitude of SR Ca release. For experiments measuring the Cai transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. In 30–40% of cells, we found that the Fura-2 transient had a bell-shaped voltage dependence. This suggests that, in these cells, the primary trigger mechanism for Ca-induced Ca-release might have been Ca entry via I Ca,L. In the remaining 60–70% of cells, the voltage dependence of the Fura-2 transient was not bell-shaped. The Fura-2 transient reached a maximum with a pulse to +10 mV, and the amplitude of the transient did not decline significantly at more positive potentials to this. In cells with a non-bell-shaped voltage dependence of the Fura-2 transient, pulses to potentials as far positive as +140 mV elicited phasic Fura-2 transients. Since this potential exceeded the Nernst potential for Ca, it was unlikely there was any tigger Ca entry via I Ca,L at this potential. This would suggest that, in these cells, another trigger for SR Ca release (in addition to I Ca,L) might be present. We conclude that rat ventricular myocytes, produced using a standard isolation technique and under standard recording conditions, can show either a bell-shaped or a sigmoidal voltage dependence of the Fura-2 transient.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050127
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  • 8
    Electronic Resource
    Electronic Resource
    Comparison of Na+-Ca2+ exchange current elicited from isolated rabbit ventricular myocytes by voltage ramp and step protocols (1999)
    Springer
    Pflügers Archiv 437 (1999), S. 944-954 
    Details
    ISSN: 1432-2013
    Keywords: Key words INa-Ca ; Na-Ca exchange ; Ventricular myocyte
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  In this study, the effects of three different voltage protocols on the Na+-Ca2+ exchange current (I Na-Ca) of rabbit right ventricular myocytes were studied. Whole-cell patch-clamp recordings were made using a Cs+-based internal dialysis solution and external solutions designed to block major interfering currents. I Na-Ca was measured at 35–37°C as (5 mM) Ni-sensitive current elicited by: a 2 s descending ramp (DR: +80 to –120 mV); a 2 s ascending ramp (AR: –120 to +80 mV) and 500 ms voltage steps (VS) between –120 and +80 mV. DR and AR were applied from –40 mV and elicited I Na-Ca with reversal potentials (E rev) of –17.6±2.5 mV (mean±SEM; n=16) and –46.2±4.1 mV (n=10; P=0.0001) respectively. This difference was maintained when the holding potential was –80 mV (–44.0±2.1 mV, n=24 and –86.3±4.8 mV, n=10; P=0.0001), when the internal Ca chelator (EGTA) was replaced with BAPTA (–19.5±1.8 mV and –46.3±1.6 mV, n=6; P=0.0003) and when DR and AR were applied alternately to the same cell. Experiments using modified ramp waveforms suggested a possible mechanism for these differences. Increases in subsarcolemmal Ca caused by Ca entry (coupled to Na extrusion) during the initial positive potential phase of the DR might have induced I Na-Ca reversal at less negative potentials than observed with AR, during the initial phase of which subsarcolemmal Ca would not have accumulated. These data suggest that I Na-Ca during voltage-clamp experiments can be significantly influenced by the type of voltage protocol chosen, as the protocol appears to induce subsarcolemmal changes in Ca and Na concentration that are independent of Ca buffering in the bulk cytosol and can occur on a pulse-to-pulse basis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050866
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