David Fedida

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.

Modulation of slow inactivation in human cardiac Kv1.5 channels by extra‐ and intracellular permeant cations

1 The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2‐ and COOH‐terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. 2 The voltage dependence of steady‐state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from ∼100% of peak current in Na+ to ∼65% in K+, ∼50% in Rb+ and ∼30% in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. 3 Changes in extracellular cation species and concentration (5‐300 mM) had only small effects on the rates of inactivation and recovery from inactivation (τrecovery∼1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. 4 Symmetrical reduction of both intra‐ and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd= 34.7 mM) and Cs+ (Kd= 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (K+1) (Kd= 43.4 mM) or Cs+1 with constant 135 mM external ion concentrations. 5 We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C‐type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter.

GATING CHARGE AND IONIC CURRENTS ASSOCIATED WITH QUINIDINE BLOCK OF HUMAN KV1.5 DELAYED RECTIFIER CHANNELS

1. The mechanism of quinidine‐induced ionic and gating current inhibition was studied in human Kv1.5 (hKv1.5) delayed rectifier channels expressed in human embryonic kidney cells. In the steady state, quinidine produced a voltage‐dependent block between +30 and +120 mV (Kd at +60 mV = 7.2 microM) with an equivalent electrical distance, delta, of 0.29 +/‐ 0.06 and 0.26 +/‐ 0.05 at 10 and 50 microM quinidine, respectively. The apparent affinity at 0 mV (Kd) was 25 microM at 10 microM quinidine and 38 microM at 50 microM quinidine. The data suggested a quinidine binding site that sensed 20‐30% of the transmembrane electrical field, from the inside. Non‐steady‐state measurements indicated rapid open channel block with mean time constants of 2.1 +/‐ 0.9 and 1.2 +/‐ 0.2 ms at 10 and 50 microM quinidine, respectively. 2. ‘On’ gating current (on‐Ig) was unaffected over a wide range of potentials and between 10 and 100 microM quinidine. On‐gating charge (Qon) was similarly conserved in the steady state between ‐100 and +50 mV. On return to ‐100 mV, quinidine slowed the off‐gating current (off‐Ig) after depolarizations more positive than ‐25 mV. After depolarizations to +50 mV, only 59 +/‐ 3.4% (10 microM quinidine) and 6.6 +/‐ 9.5% (100 microM quinidine) of the charge returned within 25 ms, compared with 100% in control. Due to the conservation of Qon in subsequent pulses, the remaining charge must have returned during the subsequent 10 s interpulse interval. 3. A threshold for quinidine action on off‐Ig was established positive to ‐25 mV. The voltage dependence of Qoff immobilization at more positive potentials than +20 mV had an equivalent electrical distance of 0.32 +/‐ 0.04 (10 microM quinidine) and 0.20 +/‐ 0.32 (100 microM quinidine) with calculated Kd values of 21.6 +/‐ 4.6 and 16.2 +/‐ 8.4 microM at 10 and 100 microM quinidine, respectively. These characteristics of block are in good agreement with values obtained from ionic data. 4. Simultaneous measurements of ionic and gating currents confirmed, after subtraction, an ionic current threshold at ‐21.8 +/‐ 1.8 mV. The gating current data confirm directly that ionic current block by quinidine occurs by binding at a site on the hKv1.5 channel that becomes accessible when the channel opens. There was no evidence for action of quinidine on kinetic states prior to the open state at concentrations of quinidine up to 100 microM.

GATING CURRENT STUDIES REVEAL BOTH INTRA- AND EXTRACELLULAR CATION MODULATION OF K+ CHANNEL DEACTIVATION

1 The presence of permeant ions can modulate the rate of gating charge return in wild‐type human heart K+ (hKv1.5) channels. Here we employ gating current measurements in a non‐conducting mutant, W472F, of the hKv1.5 channel to investigate how different cations can modulate charge return and whether the actions can be specifically localized at the internal as well as the external mouth of the channel pore. 2 Intracellular cations were effective at accelerating charge return in the sequence Cs+ > Rb+ > K+ > Na+ > NMG+. Extracellular cations accelerated charge return with the selectivity sequence Cs+ > Rb+ > Na+= NMG+. 3 Intracellular and extracellular cation actions were of relatively low affinity. The Kd for preventing slowing of the time constant of the off‐gating current decay (τoff) was 20.2 mM for intracellular Cs+ (Cs+i) and 358 mM for extracellular Cs+ (Cs+o). 4 Both intracellular and extracellular cations can regulate the rate of charge return during deactivation of hKv1.5, but intracellular cations are more effective. We suggest that ion crystal radius is an important determinant of this action, with larger ions preventing slowing more effectively. Important parallels exist with cation‐dependent modulation of slow inactivation of ionic currents in this channel. However, further experiments are required to understand the exact relationship between acceleration of charge return and the slowing of inactivation of ionic currents by cations.

Potassium Channel–Blocking Actions of Nifedipine: A Cause for Morbidity at High Doses?

To the Editor: In the last 2 years or so, the use of nifedipine, especially the short-acting form,1 2 has come under increasing scrutiny. It has been in wide use for almost two decades in the control of angina pectoris and hypertension but has been associated in a dose-dependent3 manner with unfavorable side effects like increased negative inotropy and hypotension, proarrhythmia, and in some studies, increased mortality,3 4 although this conclusion is not without controversy.5 The adverse actions of short-acting nifedipine in the acute situation in patients with hypertension1 and/or preexisting coronary heart disease are more accepted,2 and one important …

Non-specific action of methoxamine on Ito, and the cloned channels hKv 1.5 and Kv 4.2

The α1‐adrenoceptor agonist methoxamine acted independently of receptor activation to reduce Ito and the sustained outward current in rat ventricular myocytes, and hKv 1.5 and Kv 4.2 cloned K+ channel currents. Two hundred μM methoxamine reduced Ito by 36% in the presence of 2 μM prazosin, and by 37 and 38% after preincubation of myocytes with either N‐ethylmaleimide or phenoxybenzamine (n=6). The EC50 values at +60 mV for direct reduction of Ito, hKv 1.5, and Kv 4.2 by methoxamine were 239, 276, and 363 μM, respectively, with Hill coefficients of 0.87–1.5. Methoxamine accelerated Ito and Kv 4.2 current inactivation in a concentration‐ and voltage‐dependent manner. Apparent rate constants for methoxamine binding and unbinding gave Kd values in agreement with EC50 values measured from dose‐response relations. The voltage‐dependence of block supported charged methoxamine binding to a putative intracellular site that sensed ∼20% of the transmembrane electrical field. In the presence of methoxamine, deactivating Kv 4.2 tail currents displayed a distinct rising phase, and were slowed relative to control, such that tail current crossover was observed. These observations support a dominant mechanism of open channel block, although closed channel block could not be ruled out. Single‐channel data from hKv 1.5 patches revealed increased closed times with blank sweeps and decreased burst duration in the presence of drug, and a reduction of mean channel open time from 1.8 ms in control to 0.4 ms in 500 μM methoxamine. For this channel, therefore, both open and closed channel block appeared to be important mechanisms for the action of methoxamine.

Charged drug interactions at the inner mouth of K+ channels: Information gained from gating current studies

Potassium channels are integral membrane proteins which have a central role in the control of the cell environment and excitability. Gating in voltage-dependent K+ channels (Kv channels) is transduced by well-defined structural motifs that move out of the membrane in response to an applied electric field. Information about channel transitions that occur prior to opening is provided by gating currents, which reflect movement of these charged structures as transitions between kinetic closed states. Current models of gating depend heavily on information obtained from such studies. By studying modulation of the gating properties of K+ channels by cations and with drugs, a more complete interpretation of the state-dependence of drug and ion interactions with the channel can be made. In this way we can uncover the detailed mechanisms of action of K+ channel blockers such as tetraethylammonium ions and 4-aminopyridine, and antiarrhythmic agents such as nifedipine and quinidine.