Wolfhard Almers

A non‐selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions.

Membrane currents were recorded from voltage‐clamped, EGTA‐loaded muscle fibres under conditions where currents through ordinary Na+, K+ and Cl‐ channels were prevented by drugs or by absence of permeant ions (K+, and Cl‐). At 10 mM‐external [Ca2+], substitution of Na+ for the large and presumably impermeant organic cations tetramethyl‐ (TMA+) or tetraethylammonium (TEA+) failed to increase peak inward current. Hence the Ca2+ channel was not significantly permeable to Na+ under these conditions. When external [Ca2+] was reduced to levels below 1 microM in the presence of external Na+, step depolarizations to negative potentials produced tetrodotoxin‐resistant inward currents. At ‐20 mV, they rose to a peak of 30‐200 microA/cm2 within 150 ms and declined thereafter. Ca2+ and several other divalent cations reversibly blocked this inward current. The sequence of blocking potencies was Ca2+ greater than Sr2+ greater than or equal to Co2+ greater than Mn2+ congruent to Cd2+ greater than Ni2+ congruent to Mg2+. Large inward currents may be carried by Li+, Na+, K+, Rb+ and Cs+ but not by TMA+ and TEA+. The effect of external Ca2+ ([Ca2+]o) was explored over a 10(8)‐fold range in concentrations. Na+ was present at a fixed concentration. When [Ca2+]o was gradually increased from 10(‐10) to 10(‐2) M, inward current first diminished 10‐fold, reached a minimum at [Ca2+]o = 60 microM and then increased again as [Ca2+]o was increased further and Ca2+ itself became a current carrier. Block of inward current at [Ca2+]o less than 10(‐5) M could be described by binding of a single Ca2+ to a site, with a dissociation constant of the order of 0.7 microM at ‐20 mV.

Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.

We have monitored cytosolic [Ca2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Ca2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca2+], and paired stimuli reveal facilitation. Ca2+ influx through voltage-sensitive Ca2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds afer a depolarizing pulse, while they extrude or sequester the Ca2+ that has entered through Ca2+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32 degrees C, even in the absence of cytosolic K+.

Agonists that suppress M-current elicit phosphoinositide turnover and Ca2+ transients, but these events do not explain M-current suppression.

The hypothesis that acetylcholine, substance P, and LHRH suppress M-current by activating phospholipase C was tested. Each agonist caused turnover of phosphoinositide, as measured by release of inositol phosphates, and a modest transient rise in intracellular free Ca2+ ([ Ca2+]i), as determined with fura-2. Active phorbol esters depressed M-current only 50% and did not prevent further suppression by LHRH. M-current, its control by agonists, and its depression by phorbol esters were not affected by adding inositol trisphosphate or Ca2+ buffers with high or low Ca2+ to the whole-cell, voltage-clamp pipette. We conclude that phospholipase C activation does occur but does not mediate the suppression of M-current by agonists. Caffeine produced large [Ca2+]i transients and acted as an agonist to suppress M-current.

The Loose Patch Clamp

The previous chapters describe methods for recording ionic currents from very small, localized patches of membrane containing only one or at most a few channels. Many such single-channel records have to be averaged to obtain ensemble kinetics or current densities through the membrane. An alternative would be to record from many channels simultaneously, using patch pipettes for a whole-cell recording (Hamill et al., 1981; Chapter 7) or the classic voltage-clamp techniques, with loss of the positional information. Also, the “gigaseals” needed for single-channel recording require extensive proteolytic enzymatic treatment on many cells in order to clean the membrane surface.

Voltage clamp of rat and human skeletal muscle: measurements with an improved loose-patch technique.

Intact fibres of human intercostal and rat omohyoid muscles were studied at 23 degree C with a loose‐patch voltage‐clamp technique that employed two concentric micropipettes to electrically isolate small‐diameter (10‐15 microns) patches of sarcolemma. This method allows investigation of membrane excitability under highly physiological conditions. Step depolarizations to 0 mV elicited sodium inward currents that reached peak values of up to 20 mA/cm2 within 250 microseconds, and then declined. In human muscle, the reversal potential (ENa) was approximately 40 mV, and maximal conductances (GNa) ranged from 44 to 360 mS/cm2. In rat muscle, ENa was 42 mV and GNa ranged from 100 to 250 mS/cm2. Sodium channels in rat and human muscle were indistinguishable in most aspects of their kinetic behaviour and voltage dependence. Outward potassium currents were small by comparison (usually less than 2 mA/cm2) and saturated at positive potentials. The maximum potassium conductance (GK) ranged from 0 to 19 mS/cm2 (human) and from 4 to 12 mS/cm2 (rat muscle).

Slow calcium and potassium currents in frog skeletal muscle: their relationship and pharmacologic properties

Slow Ca and K currents across frog skeletal muscle membrane were recorded with the Vaseline gap voltage clamp in order to investigate block by divalent cations and various organic compounds. Cd2+, Ni2+, Co2+, Mn2+, Mg2+ all block Ca currents, as do barbiturates, D-600 and nifedipine. Local anesthetics also block Ca currents, with the impermeant quaternary lidocaine derivative, QX-314, being more than an order of magnitude less potent than its permeant parent compound. Surprisingly, all agents that blocked Ca currents also blocked the slow K currents. To explain this pharmacologic parallel, one could suggest that K current is activated by Ca2+ appearing in the myoplasm due to the combination of Ca current and release from internal stores. While possibly correct for intact fibres, this hypothesis appears not to apply in our case where the myoplasm contained the Ca chelator EGTA at high concentration. Instead, K currents seem to be activated by a decrease in external [Ca2+]. In the transverse tubules, Ca current is known to cause [Ca2+] to decline to submicromolar concentrations, and evidence is presented that K currents are activated by Ca depletion from a restricted extracellular space. It is suggested that K currents flow through Ca channels that have become capable of passing monovalent cations after the tubules have become depleted of Ca2+.

Patch voltage clamping with low-resistance seals : loose patch clamp

Publisher Summary This chapter discusses several loose-seal methods, and emphasizes the possible difficulties associated with each, describing how they can be avoided. The chapter also discusses applications in which the potential across the membrane is controlled. The basic loose-patch clamp, which employs a single loosely sealed extracellular pipette and no intracellular electrodes, is appropriate for studying rapidly activating currents, such as voltage-gated Na+ and K+ currents, in muscles and other large cells with stable resting potentials. This technique is not well suited to studying currents that are much smaller or slower, such as the voltage-gated Ca2+ current in skeletal muscles, because of artifacts associated with the low resistances of the loose seals. A combination of loose-seal patch recordings, tight-seal whole-cell recordings, and freeze-fracture electron micrographs indicate that the array of membrane particles seen at presynaptic active zones on hair cells are dusters of ion channels that contain a carefully regulated mixture of voltage-gated Ca2+ channels and Ca2+-activated K+ channels. Future uses of the loose-seal technique are likely to address a wider range of problems, such as local mechanisms of channel modulation that require preservation of the microscopic structure of the membrane or its spatial relationship to the cytoskeleton.

Effect of glucocorticoid treatment on the excitability of rat skeletal muscle

Dexamethasone treatment in the rat produced depolarization of extensor digitorum longus (EDL) muscle fibers but not soleus (SOL) fibers studies in vitro at 23° C. The depolarization of EDL fibers was most prominent after 1 day of treatment (treated−77.5±1.1 mV, control−87.2±0.8 mV; mean±S.E.), and was associated with elevation of the action potential threshold and reduction of the action potential overshoot. In vivo, or in vitro in chloride-free solution, the resting potential and action potential threshold and overshoot of EDL fibers from glucocorticoid-treated and control rats were similar. Sodium currents were studied with a patch voltage clamp. Glucocorticoid treatment did not alter the voltage dependence of sodium channel activation or inactivation currents at about −29 mV and half-maximal inward currented at about −50 mV. Sodium channels were half inactivated at about −71 mV. Glucocorticoid treatment did not alter the sarcolemmal resistance or capacitance. We conclude that glucocorticoid treatment does not produce muscle weakness or atrophy by altering the excitability of muscle fibers.

An improved loose patch voltage clamp method using concentric pipettes

A method for noninvasive voltage-clamp recording from large cells is described. A firepolished pipette having two concentric barrels is pushed against the cell membrane, thereby electrically isolating a circular patch subdivided into an inner and an annular outer region. Both regions are held isopotential, but current is collected from the inner region only. The method electrically simulates a high resistance seal between pipette and cell membrane, allowing accurate and rapid voltage-clamp recording under conditions where the seal resistances actually obtained are low (near 1 MΩ). This is useful in applications where one wishes to avoid enzymatic treatment.We provide details of electrode construction and voltage-clamp electronics, and present results obtained from frog skeletal muscle and leech neurons. For sodium channels of frog muscle, extensive data were previously obtained with other methods. There is good agreement between the earlier results and the measurements presented here.

Exocytosis and its control at the synapse

Several new approaches have given fresh insight into the mechanism and control of exocytosis. Electrophysiological and morphological studies show that many or all of the intramembrane particles at presynaptic active zones are voltage-gated Ca2+ channels. The sensitivity and time resolution of voltammetry allow the time course with which a single vesicle releases transmitter to be studied. Membrane proteins of the cell surface and synaptic vesicles have been shown to interact, and may join to form the fusion-pore complex.