Chloride current in toad skeletal muscle and its modification by the histidine-modifying reagent diethylpyrocarbonate.

Cl- currents were measured in short fibres in the toad lumbricalis muscle with a two-microelectrode voltage clamp. Membrane Cl- conductance increased markedly when external pH was raised. At pH 7 or higher, the Cl- current fell during a hyperpolarizing voltage pulse and the rate of inactivation was directly proportional to the voltage change. The histidinemodifying reagent diethylpyrocarbonate (DEPC, 1 mM) which carbethoxylates histidil residues in proteins, suppressed the inactivation of Cl- currents at pH 7.5. On the other hand, no apparent changes in the kinetics of the currents at pH 5 were seen. No3- currents, which are independent of the extracellular pH and time, were not affected by DEPC. Our results support the notion that the inactivation of Cl- currents at pH 7.5 represents a membrane permeability change and that DEPC interferes with this process. Protonation of histidine groups associated with Cl- channels may be the controlling reaction for the pH -dependent Cl- response.

Blockade of the inward rectifier potassium currents by zinc and nickel ions in voltage-clamped toad muscles.

The inward rectifier is one of the voltage-sensitive K+ channels present in several tissues: Its conductance increases under hyperpolarization and decreases with depolarization. In this work we studied the effects of Zn2+ and Ni2+ (5-30 mM) on the macroscopic K+ current through the inward rectifier system. The experiments were performed in the short muscle fibers of the lumbricalis muscle of toads with a two-microelectrode voltage clamp technique. The fibers were equilibrated in a control solution containing 68 mM K2SO4 and then exposed to Zn2+ or Ni2+. We found that both cations reduced in a reversible manner the current carried by K+ ions, and this reduction was prevented by decreasing the external pH of the solution (pH 5). The blockade of current was slightly dependent on the membrane potential and time independent. Two mechanisms may be involved in the blocking action of these cations: Zn2+ and Ni2+ may either be blocking the pore of the channels or acting at a regulatory binding site on the extracellular surface in an unspecified manner.

Barium resistant potassium current in mammalian skeletal muscle following denervation.

Inward rectifier potassium channels are thought to be related to resting membrane potential and in innervated skeletal muscle they are specially sensitive to the blocking action of Ba2+ ions. After denervation other channels are known to become resistant to their blockers. We study the effect of Ba2+ upon the inward rectifier potassium channels after denervation. Rat extensor digitorum longus fibers were equilibrated for 150 minutes in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 35 minutes. This repolarization was blocked by 5 mM BaCl2 in innervated muscles and in muscles denervated for 7 days, but failed to do so after 14 days of denervation. Voltage-clamp experiments performed in lumbricalis denervated muscle showed a lack of effect of Ba2+ upon potassium current after 18 days of denervation. This results suggest that the inward rectifier potassium channels become resistant to Ba2+ ions after denervation, indicating a neural influence.

Effect of electrode impedance in improved buffer amplifier for bioelectric recordings.

We analysed the effects of electrode impedance on the transfer response of a one-stage improved buffer amplifier. The electrode DC resistance (R(d)) modifies the one-stage buffer transfer response. We found a limit electrode resistance (R(d(lim))) which depends on the transfer damping factor (epsilon). If R(d) is lower than 86.5 komega, the transfer response of the buffer fulfils American Heart Association (AHA) recommendations, but when R(d) is greater than R(d(lim)) it must be cautiously weighed up because its influence in the transfer response becomes appreciable. The maximum R(d) that can be driven by the buffer is 1.2 Momega. Higher values do not fulfil AHA recommendations. Therefore, electrodes with higher impedance should not be used with this kind of buffer. In contrast, when this buffer is used to build in an instrumentation amplifier (IA) for bipolar recording, the common-mode rejection ratio (CMRR) is sensitive to the electrode type used.

[Ion channels in non excitable cells]. / Canales iónicos en células no excitables.

Several distinct types of voltage-gated and second-messenger-operated K+, Ca2+, Na+ and Cl- channels exist in electrically non excitable cells such as those of the hematopoietic lineage. In these cells ion channels mediate cellular functions involving intracellular biochemical responses, rather than rapid electrical signaling. The presence of the channels is required for several basic functions, such as activation, secretion of lymphokines, mitogenesis, the regulation of cell volume and the mechanisms of resistance to chemotherapeutic agents. Here IN we review the patch-clamp method for studying many characteristics of these ionic channels, particularly in blood cells.

Chloride channels in toad skeletal muscle fibers.

Chloride currents were measured in short lumbricalis fibers of toads (Bufo arenarum) with voltage and patch clamp techniques. For the availability of chloride currents we applied a double-pulse technique in voltage-clamped fibers. When the test pulse was preceded by a positive prepulse, the initial current was larger than with a negative prepulse and exhibited a different rate of decline to its steady-state value. At the single-channel level we found that in most of the experiments with symmetrical 110 mM NaCl solutions, two levels of conductance, 20 ("small channel") and 360 pS ("maxi channel"), occurred with the highest probabilities. The openings of the maxi channels were more frequent at potentials close to 0 mV, whereas for the small channels the openings were at negative potentials. In contrast with the results with the macroscopic currents, a change of 2 orders of magnitude in the pH, from 7.3 to 5, had only minor effects on the channels' conductance. As with some other anion channels, the selectivity of the channels described here is low, the p(Cl)/p(Na) ratio being 1.9 and 3.7 for the small and maxi Cl(-) channels, respectively. The behavior of these Cl(-) channels with a relative high Na(+) permeability could contribute to the relatively low resting membrane potential of the lumbricalis fibers measured in the standard 110 mM NaCl solution.

Effects of amiodarone and desethylamiodarone on the inward rectifying potassium current (IK1) in rabbit ventricular myocytes.

We examined the effects of amiodarone (AMI) and desethylamiodarone (DAM) on whole-cell inward rectifying potassium current (IK1) in freshly isolated adult rabbit ventricular myocytes by using the whole-cell voltage-clamp technique, as an index of their effects on resting membrane resistance (Rm). Under control conditions, the current showed a strong inward rectification with a maximal inward current measured at -130 mV of -26.4 +/- 1.3 pA/pF and a maximal outward current measured at -50 mV of 3.5 +/- 0.3 pA/pF The current also exhibit a time-dependent activation, with a time constant of activation (tau(a)) that increased with depolarization. The maximal slope conductance normalized to cell capacitance was 0.509 +/- 0.019 nS/pE After exposure to both DAM (50 microM; n = 8) and AMI (50 microM; n = 7), rapid decrease in inward IK1 was observed. Block was restricted almost exclusively to the inward component. DAM caused a significant reduction of the maximal inward current (-20.0 +/- 2.0 pA/pF; p < 0.05), whereas AMI induced an even greater reduction of the same component (-14.1 +/- 1.2 pA/pF; p < 0.05 with respect to control and to DAM). The outward component of IK1 was not changed by either AMI or DAM (4.0 +/- 0.3 pA/pF and 3.4 +/- 0.4 pA/pF, respectively). AMI and DAM also decreased the maximal slope conductance significantly (0.297 +/- 0.019 nS/pF and 0.421 +/- 0.038 nS/pF, respectively). In addition, AMI but not DAM significantly increased the tau(a). However, the voltage dependence of the acceleration of tau(a) remained unchanged after both AMI and DAM exposure. These results allow us to conclude that AMI may induce a greater increase in the resting Rm than its main metabolite. This effect may counterbalance, at least in part, the conduction slowing due to its sodium channel-blocking properties.

Differential modulation of electrocardiographic indices of ventricular repolarization dispersion depending on the site of pacing during premature stimulation.

INTRODUCTION: Dispersion of ventricular repolarization has been shown to increase with premature stimulation. Moreover, a straight correlation between the amount of dispersion of repolarization and the vulnerability to ventricular fibrillation was reported. On the other hand, differences between right ventricular (RV) and left ventricular (LV) fibrillation threshold have been reported. However, no data exist regarding the influence of the site of stimulation on modulation of dispersion of repolarization. METHODS AND RESULTS: In the present study, several ECG indices of dispersion of repolarization, as a function of the coupling interval and the site of stimulation, were evaluated in a modified Langendorff-perfused rabbit heart (n = 12), with a 5 x 8 array of a simulated body surface unipolar lead system. As the coupling interval was shortened, a biphasic modulation of dispersion of repolarization was found when stimuli were elicited at the LV. In contrast, when the heart was paced from the RV, the dispersion increased monotonically as coupling interval was shortened. CONCLUSION: A differential behavior of the modulation of dispersion of repolarization was found as a function of the site of stimulation.

Influence of filtering techniques on the time-domain analysis of signal-averaged P wave electrocardiogram.

INTRODUCTION: The advent of signal-averaged ECG (SAECG) systems for P wave analysis has made it important to determine if the use of different filtering techniques in these systems is diagnostically equivalent. METHODS AND RESULTS: Three different high-pass filtering techniques and two cutoff frequency values were used: 29- and 40-Hz Butterworth bidirectional filter (BB29, BB40), 29- and 40-Hz Butterworth unidirectional filter (UB29, UB40), and 29- and 40-Hz least mean square filter (LMS29, LMS40). Normal healthy volunteers (n = 36) and patients with documented paroxysmal atrial fibrillation (n = 23) were analyzed. A custom-built SAECG system and standard bipolar orthogonal leads were used. Noise was reduced to < 0.3 microV. P wave total duration, root mean square voltage of the terminal 20, 30, and 40 msec of the filtered vector magnitude, and the area under the curve between the onset and offset of averaged unfiltered and filtered P wave vector magnitude were analyzed. Only the duration of the P wave showed statistically significant differences between groups, being longer in the PAF group for all filters and cutoff frequencies studied. A bias increment of approximately 20 msec was detected in unidirectional and least mean square filters as compared to the bidirectional filter. Sensitivity, specificity, and predictive accuracy were > 70% for all filters; the BB40 filter yielded the best performance. CONCLUSION: The normality limits derived from one filter cannot be applied directly to recordings obtained from the other filters. Critical limits must be established individually for different software settings.