Enrico Stefani

Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels

In voltage-dependent ion channels, a voltage sensor region is responsible for channel activation and an aqueous pore is responsible for ion conduction. These two processes have been traditionally considered to be independent. We describe here a mutation in the putative pore region (W434F) that completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the nonconductive mutant were found to be identical in their kinetic and steady-state properties to those in conductive channels. Gating current measurements could be performed without subtracting pulses and in the presence of normal physiological solutions. Application of internal tetraethylammonium (an open channel blocker) induced Off charge immobilization for large depolarizations, suggesting that the internal tetraethylammonium-binding site becomes available upon depolarization. We concluded that for this mutant, although the conduction pathway is not functional, the channel can still undergo the closed-open conformation in response to voltage changes.

Serum Antibodies to L-Type Calcium Channels in Patients with Amyotrophic Lateral Sclerosis

BACKGROUND AND METHODS Sporadic amyotrophic lateral sclerosis is a chronic, progressive degenerative disease of the motor neurons of the spinal cord and motor cortex. The cause is unknown. Recent electrophysiologic studies in animals indicate that immunoglobulins from patients with this disease alter presynaptic voltage-dependent calcium currents and calcium-dependent release of neurotransmitters. To determine whether similar interactions might be identified biochemically, we used an enzyme-linked immunosorbent assay (ELISA) to detect the reaction of serum IgG with purified complexes of L-type voltage-gated calcium channels from rabbit skeletal muscle. The results from patients with amyotrophic lateral sclerosis were compared with those obtained from patients with other types of motor neuron disease, patients with autoimmune and non-autoimmune neurologic diseases, and normal subjects. RESULTS Serum samples from 36 of 48 patients with sporadic amyotrophic lateral sclerosis (75 percent) contained IgG that reacted with L-type calcium-channel protein, and serum reactivity on ELISA correlated with the rate of disease progression (Spearman rank-correlation coefficient, 0.62). Reactive serum was present in only 1 of 25 normal subjects and 1 of 35 control patients with no motor neuron disease. Antibodies to L-type voltage-gated calcium channels were identified in 6 of 9 patients with Lambert-Eaton syndrome, and in 3 of 15 patients with Guillain-Barré syndrome. CONCLUSIONS Antibodies to L-type voltage-gated calcium channels are present in the serum of patients with amyotrophic lateral sclerosis, and antibody titers correlate with the rate of disease progression. Together with previous data, these results suggest a role for autoimmune mechanisms in the pathogenesis of sporadic amyotrophic lateral sclerosis.

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).

Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.

Evidence for autoimmunity in amyotrophic lateral sclerosis

Although the etiology and pathogenesis of ALS is unknown, increasing evidence supports a role for autoimmune mechanisms in motoneuron degeneration and death. An animal model, experimental autoimmune gray matter disease, can be induced by the inoculation of spinal cord gray matter. The experimental disease is characterized by weakness secondary to the loss of upper and lower motoneurons, accompanied by inflammatory foci within the spinal cord, and IgG at the neuromuscular junction and within UMN and LMN. In human ALS, IgG is present within the UMN and LMN, and T-lymphocytes and activated microglia have been identified within spinal cord gray matter and motor cortex. ALS IgG can passively transfer physiological changes of the neuromuscular junction to mice resulting in enhanced release of acetylcholine. The ALS IgG selectively interact with calcium channels and alter channel function. These data suggest a potential role for autoimmune mechanisms in the destruction and loss of motoneurons in ALS.

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.

Calcium transients in single mammalian skeletal muscle fibres.

1. We studied the transient changes in myoplasmic Ca2+ concentration under current‐ and voltage‐clamp (double Vaseline‐gap technique) in cut fibres of rat extensor digitorum longus muscle using mag‐fura‐2 (furaptra) as Ca2+ indicator, at 3.6‐3.8 microns sarcomere length and 17 degrees C. Mag‐fura‐5 and fura‐2 were also used in order to characterize some aspects of the Ca2+ transients. 2. The peak [Ca2+] in response to a single action potential was 4.6 +/‐ 0.4 microM (n = 5). The time to peak of the Ca2+ transient was 4.6 +/‐ 0.42 ms, with half‐width of 8.2 +/‐ 1.5 ms, time constant of the rising phase 1.15 +/‐ 0.25 ms, time constant of the decaying phase 3.26 +/‐ 0.65 ms, and delay between action potential and Ca2+ transient 2.0 +/‐ 0.2 ms. 3. Ca2+ transients were studied under voltage‐clamp conditions at different voltages and pulse durations. The rising phase showed a complex temporal course with a fast initial increase and a second component. Both components were separated by a plateau or a brief decrease of the Ca2+ concentration. The peak Ca2+ transient was 10.5 +/‐ 1.3 microM (n = 22). 4. After interrupting the pulse, Ca2+ concentration decayed exponentially. The time constant of decay of the Ca2+ transient increased with the pulse voltage and duration, reaching a maximum value at potentials more positive than +10 mV and pulses longer than 200 ms. An analysis of the decaying phases of the Ca2+ transients suggests that only the removal process operates after fibre repolarization. 5. The rate of Ca2+ release from the sarcoplasmic reticulum was calculated using the Melzer, Ríos & Schneider model. The value of 17.2 +/‐ 3.1 micronM ms‐1 (n = 10) estimated in these calculations was intermediate between those obtained by other authors from cut frog muscles (10 microM ms‐1) and intact frog fibres (100 microM ms‐1) using antipyrylazo III (AP III) as the Ca2+ indicator.

Calcium current and charge movement of mammalian muscle: action of amyotrophic lateral sclerosis immunoglobulins.

1. The Vaseline‐gap voltage clamp technique was used to record dihydropyridine (DHP)‐sensitive Ca2+ currents (ICa) and charge movement in single cut fibres from the rat extensor digitorum longus (EDL) muscle. Amyotrophic lateral sclerosis (ALS) immunoglobulin G (IgG) action on ICa and charge movement has been characterized. 2. ALS IgG reduced ICa amplitude. The peak ICa of EDL fibres (mean +/‐ S.E.M.) at 0 mV, expressed as amperes per membrane capacitance, was ‐4.79 +/‐ 0.029 A F‐1, while after 30 min incubation in ALS IgG it was ‐2.52 +/‐ 0.04 A F‐1. IgG from healthy patients, and from patients with other diseases (familial ALS, myasthenia gravis, chronic relapsing inflammatory polyneuritis, multiple sclerosis and one sample from Lambert‐Eaton syndrome, LES) did not affect ICa, while IgG from patients with Guillain‐Barré syndrome and one other sample from a patient with LES affected the ICa in a similar way as ALS IgG. 3. The time constant of ICa activation (alpha m) at 0 mV was 44.8 +/‐ 1.4 ms in control, and 36.6 +/‐ 1.5 ms after an incubation of 30 min in ALS IgG. The steady‐state activation curve (m infinity) was shifted to more positive potentials by ALS IgG. 4. The rate constants of activation (range ‐20 to 30 mV) were altered by ALS IgG: alpha m decreased while beta m increased. These data suggest that ALS IgG favours the permanence of the Ca2+ channels in the closed state. 5. The time constant of Ca2+ channels deactivation at ‐90 mV with a pre‐pulse to 0 mV was 4.4 +/‐ 0.5 ms in control and 4.1 +/‐ 0.6 ms in ALS IgG. The relationship between the deactivation time constant and membrane potential was not significantly modified by ALS IgG. 6. ICa inactivation was not affected by ALS IgG. The potentials of half‐inactivation were ‐32.1 and ‐36.6 mV in control and ALS IgG, respectively. Similarly, the rate constants of inactivation (alpha h and beta h) remained unaltered by ALS IgG. 7. We successfully blocked ICa with 100 microM‐TMB‐8 (3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)octyl ester hydrochloride), without major effects on charge movement. We adopted this procedure to study charge movement. ALS IgG reduced charge movement without significant effects on the effective valence and voltage dependence. Qon and Qoff, the charges during and after the pulse, were similarly affected by ALS IgG. 8. The actions of ALS IgG on DHP‐sensitive Ca2+ current and charge movement suggest an interaction between ALS IgG and some component of the DHP‐receptor complex.

The action of amyotrophic lateral sclerosis immunoglobulins on mammalian single skeletal muscle Ca2+ channels.

1. The planar phospholipid bilayer technique was used to study the T‐tubule skeletal muscle dihydropyridine (DHP)‐sensitive calcium (Ca2+) channel. To improve the signal‐to‐noise ratio, Ca2+ channel activity was recorded using both 800‐50 and 500‐50 mM NaCl gradients. 2. Ca2+ channels were characterized by their cation selectivity and pharmacological profile. The mean open time for channels identified by these techniques was increased by the DHP agonist Bay K 8644 (2 microM), while it was decreased by the DHP antagonist nifedipine (5 microM). Nifedipine also reduced Ca2+ channel amplitude levels. 3. Immunoglobulins G (IgG) from three amyotrophic lateral sclerosis (ALS) patients (n = 14 experiments), one myasthenia gravis (MG) patient (n = 3 experiments) and one healthy individual (n = 4 experiments), were tested on Ca2+ channel activity at a final concentration of 3 mg/ml. 4. Channel mean open time, mean closed time and time integral for the current were not modified by normal IgG (n = 4 experiments). Similarly, MG IgG did not reduce channel activity (n = 3 experiments). 5. ALS IgG reduced the mean open time of DHP‐sensitive Ca2+ channel activity in twelve out of fourteen experiments. In addition, in five out of twelve experiments, ALS IgG stabilized the channel to a smaller amplitude level. 6. ALS IgG reduced Ca2+ channel activity in a side‐selective fashion, probably corresponding to the external side of the channel. 7. These results suggest that ALS IgG action on DHP‐sensitive Ca2+ channels is not mediated by second messengers, thus favouring a direct mechanism for interaction with the DHP receptor complex.

Ca2+ current and charge movement in adult single human skeletal muscle fibres.

1. The Vaseline‐gap technique was used to record calcium currents (ICa) and charge movement in single cut fibres from normal human muscle. Experiments were carried out in 2 or 10 mM‐extracellular Ca2+ concentration ([Ca2+]o) and at 17 or 27 degrees C. 2. The passive electrical properties of the fibres with this technique were: membrane resistance for unit length rm = 59.4 k omega cm; longitudinal resistance per unit length ri = 4.9 M omega/cm; longitudinal resistance per unit length under the Vaseline seals re = 438 M omega/cm; specific membrane resistance Rm = 1.176 k omega cm2; input capacitance = 5.53 nF; specific membrane capacitance = 8.9 microF/cm2. 3. The maximum amplitude of ICa at 17 degrees C was: in 2 mM [Ca2+]o, ‐0.42 microA/microF and in 10 mM [Ca2+]o, ‐1.44 microA/microF. At 27 degrees C and in 10 mM [Ca2+]o, it increased to ‐3.04 microA/microF. The calculated temperature coefficient (Q10) for the increase in amplitude from 17 to 27 degrees C was 2.1. 4. Ca2+ permeability (PCa) was calculated using the Goldman‐Katz relation; in 2 mM [Ca2+]o at 17 degrees C, PCa = 1.26 x 10(‐6) cm/s; in 10 mM [Ca2+]o at 17 degrees C, PCa = 2.23 x 10(‐6) cm/s; in 10 mM [Ca2+]o at 27 degrees C, PCa = 4.03 x 10(‐6) cm/s. 5. The activation curve calculated from the PCa was shifted by 10 mV to positive potentials when raising [Ca2+]o from 2 to 10 mM. Increasing the temperature did not change the curve. The mid‐point potentials (Va 1/2) and steepness (k) of the activation curves were: at 17 degrees C, in 2 mM [Ca2+]o, Va 1/2 = ‐1.53 mV and k = 6.7 mV; in 10 mM [Ca2+]o, Va 1/2 = 9.96 mV and k = 6.8 mV; at 27 degrees C and 10 mM [Ca2+]o, Va 1/2 = 11.3 mV and k = 7.7 mV. The activation time constant in 10 mM [Ca2+]o reached a plateau at potentials positive to 10 mV, with a value of 93.8 ms at 17 degrees C and 17.4 ms at 27 degrees C. The calculated Q10 was 4.5. 6. The deactivation of the current was studied from tail currents at different membrane potentials in 10 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)