Retention in the endoplasmic reticulum as a mechanism of dominant-negative current suppression in human long QT syndrome.

Mutations in the cardiac potassium channel HERG (KCNH2) cause chromosome 7-linked long QT syndrome (LQT2) characterized by a prolonged QT interval, recurrent syncope and sudden cardiac death. Most mutations in HERG exhibit "loss of function" phenotypes with defective channels either inserted into the plasma membrane or retained in the endoplasmic reticulum. "Loss of function" mutations reduce I(Kr), the cardiac delayed rectifier current encoded by HERG, due to haploinsufficiency or suppression of wild-type function by a dominant-negative mechanism. One explanation for dominant-negative current suppression is that mutant subunits render tetrameric channel complexes non-conducting on co-assembly. In the present paper we describe an alternative mechanism for this phenomenon. We show (1) that the dominant-negative HERG mutation A561V is retained in the endoplasmic reticulum and (2) that wild-type channels are tagged for retention in the ER by co-assembly with trafficking deficient A561V subunits. Thus, in HERG A561V dominant-negative suppression of wild-type function is the result of an acquired trafficking defect.

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells.

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

Effects of dexamethasone on L-type calcium currents in the A7r5 smooth muscle-derived cell line.

Patch clamp experiments were used to characterize the effect of dexamethasone on calcium currents in A7r5 cells. Pretreatment for 48 h with 200 nM dexamethasone did not affect the single channel conductance, the voltage dependence of channel opening, or the voltage-dependent inactivation of L-type channels. However, dexamethasone caused an approximately 2-fold increase in the amplitude of L-type calcium currents in 5 out of 9 experiments, suggesting an increase in the number of active channels. The effect of dexamethasone appeared to be greatest on batches of cells with low control current density. The amplitude of T-type calcium current was not affected by dexamethasone.

Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.

Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes.

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca2+ to provide the simultaneous activation of the (Ca2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 degrees C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.

Effects of resting membrane potential and intactness of the T-tubules on caffeine contractures in rat skeletal muscle.

We have studied the effects of changes in the resting membrane potential (Vm) and T-tubules on caffeine contracture (25 mM) elicited in rat soleus muscle in vitro at 34 degrees C. In high [K]o (30-140 mM, [K]o X [Cl]o constant) caffeine contractures were reduced by about 40-50% and had a faster time course than in normal Krebs ([K]o = 5 mM). Detubulation of the muscles by an osmotic treatment produces a reduction of about 30% in the caffeine contracture tension. Our results with high K solutions suggest a reduced sensitivity of the myofibrils to calcium released by caffeine. The effects of detubulation on caffeine contracture suggest that caffeine may have a direct effect on sarcolemma in addition to its well known action on the sarcoplasmic reticulum (SR). However, a depletion of the calcium content in the SR of depolarized muscle fibres as well as an anatomical damage produced by the osmotic treatment can not be ruled out as an explanation for the reduced caffeine contracture.

Caffeine contractures in denervated slow-twitch and fast-twitch muscles of the rat.

Caffeine contractures (25 mM) and twitches were registered in vitro (34 degrees C) in normal and denervated soleus and extensor digitorum longus of the rat. Both muscles lost weight progressively after denervation although the loss of weight in soleus muscles was more manifest. Denervated extensor digitorum longus muscles showed an increase in the caffeine contracture tension (expressed in g/g muscle) whereas the response generated by denervated soleus muscles was smaller than the control values. The time to peak of the caffeine responses was shortened in both types of muscles after denervation. Twitches were larger and had a slower time course than in normal muscles, especially in the extensor digitorum longus muscles. These findings suggest that some changes in the excitation-contraction coupling could be responsible for the potentiation of the twitches in denervated muscles.

The effect of verapamil and Ca free solution on mechanical and electrical properties in fast twitch mammalian skeletal muscle.

The effects of verapamil (0.01-0.1 mM) and Ca-free solutions (0 Ca, 3 mM MgCl2, 5 mM EGTA) upon mechanical and electrical properties of a fast twitch mammalian skeletal muscle (extensor digitorum longus) have been studied. Ca-free saline reduces twitch and tetanus tension. The response to single pulses was not affected in the presence of verapamil, but as the rate of stimulation increased, peak tensions were lower and a gradual decrease of tension was observed: frequency-dependent effect. Caffeine (30 mM) contracture was significantly reduced by both solutions. The fibers in Ca-free saline were depolarized (6 mV) and +dV/dt, -dV/dt and overshoot of the AP's were reduced. On the other hand, verapamil did not affect Vm. The presence of 0.1 mM verapamil hindered the ability of the fibers to generate AP's at high rates of stimulation. Lower concentration of verapamil (0.01) did not affect the repetitive electrical activity, but tetanic tension was decreased. These findings support partially the hypothesis about the dependence of mammalian skeletal muscle from external Ca. Verapamil would have several mechanisms of action: a) Ca-channel blocker action, b) local anaesthetic effect, and c) reduction of Ca release from the sarcoplasmic reticulum.

The effect of verapamil and Ca free solution on mechanical and electrical properties in fast twitch mammalian skeletal muscle.

The effects of verapamil (0.01-0.1 mM) and Ca-free solutions (0 Ca, 3 mM MgCl2, 5 mM EGTA) upon mechanical and electrical properties of a fast twitch mammalian skeletal muscle (extensor digitorum longus) have been studied. Ca-free saline reduces twitch and tetanus tension. The response to single pulses was not affected in the presence of verapamil, but as the rate of stimulation increased, peak tensions were lower and a gradual decrease of tension was observed: frequency-dependent effect. Caffeine (30 mM) contracture was significantly reduced by both solutions. The fibers in Ca-free saline were depolarized (6 mV) and +dV/dt, -dV/dt and overshoot of the APs were reduced. On the other hand, verapamil did not affect Vm. The presence of 0.1 mM verapamil hindered the ability of the fibers to generate APs at high rates of stimulation. Lower concentration of verapamil (0.01) did not affect the repetitive electrical activity, but tetanic tension was decreased. These findings support partially the hypothesis about the dependence of mammalian skeletal muscle from external Ca. Verapamil would have several mechanisms of action: a) Ca-channel blocker action, b) local anaesthetic effect, and c) reduction of Ca release from the sarcoplasmic reticulum.

Effects of actinomycin D on contractile properties of denervated rat skeletal muscle.

We studied the mechanical and electrical properties in the extensor digitorum longus muscle of Wistar rats in vitro 4 days after denervation. In a group of animals, actinomycin D was injected intravenously at the time of denervation. The prolongation of contraction and relaxation time, and the increase in twitch tension observed in denervated muscles were prevented by the drug. Other denervatory changes such as the elimination of cooling potentiation, posttetanic potentiation, staircase phenomenon, and shortening of caffeine contracture were also inhibited or partially reverted by actinomycin D. In addition, some electrical denervatory changes such as the decrease of resting membrane potential and slowing of maximum rate of fall of the action potential were partially abolished by actinomycin D. These results support the hypothesis that a new synthesis of proteins is related to denervation changes.

Co2+, low Ca2+, and verapamil reduce mechanical activity in rat skeletal muscles.

We have studied the effects of Co2+ (5 mM), low-Ca2+ solution [0 added CaCl2, 5 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, 3 mM MgCl2 Ringer], and verapamil (0.1 mM) on mechanical and electrical properties of rat soleus muscle in vitro at 34 degrees C. Muscle fibers had normal resting potentials in Co2+ and verapamil solutions. Low-Ca2+ solution produces a depolarization of approximately 4 mV. The action potentials are smaller and have a slower time course when exposed to test solutions. Iterative generation of action potentials in the presence of Co2+ and low-Ca2+ solution is not modified. In the presence of Co2+ or low-Ca2+ solution, the mechanical output, twitch and tetanus tensions, and caffeine contracture are reduced significantly. Verapamil produces a decrease in the twitch and tetanic tensions but does not modify the caffeine contracture tension. The effect of verapamil on the twitch becomes more manifest when the muscle is stimulated at 3-5 Hz. We suggest that changes in the action potential characteristics or the inhibition of a Ca2+ current are responsible for the mechanical changes observed in the presence of the drugs.