Faculty Bibliography

Summary of a symposium presented by the American Physiological Society (Cell and General Physiology Section and Muscle Group) at the 70th Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, Missouri, April 15, 1986, chaired by M. Lieberman and F. Fay. This symposium reflects a growing interest in seeking new technologies to study the basic physiological and biophysical properties of cardiac, smooth, and skeletal muscle cells. Recognizing that technical and analytical problems associated with multicellular preparations limit the physiological significance of many experiments, investigators have increasingly focused on efforts to isolate single, functional embryonic, and adult muscle cells. Progress in obtaining physiologically relevant preparations has been both rapid and significant even though problems regarding cell purification and viability are not fully resolved. The symposium draws attention to a broad, though incomplete, range of studies using isolated or cultured muscle cells. Based on the following reports, investigators should be convinced that a variety of experiments can be designed with preparations of isolated cells and those in tissue culture to resolve questions about fundamental physiological properties of muscle cells

Blockade of Ca2+ channels by omega-conotoxin GVIA, a 27 amino acid peptide from the venom of the marine snail Conus geographus, was investigated with patch-clamp recordings of whole-cell and unitary currents in a variety of cell types. In dorsal root ganglion neurons, the toxin produces persistent block of L- and N-type Ca2+ channels but only transiently inhibits T-type channels. Its actions appear to be neuron-specific, since it blocks high-threshold Ca2+ channels in sensory, sympathetic, and hippocampal neurons of vertebrates but not in cardiac, skeletal, or smooth muscle cells. Block occurs through direct interaction of the toxin with an external site closely associated with the Ca2+ channel, without apparent involvement of a second messenger or dependence on channel gating. The tissue and channel-type specificity and the directness and slow reversibility of the block are features that favor use of omega-conotoxin as a tool for purifying particular neuronal Ca2+ channels and defining their physiological function

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell

We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between 'permeators' and 'blockers,' only quantitative differences in how quickly ions enter and leave the pore

Ca2+ channels allow passage of Ca2+ ions into the cytoplasm through a selective pore which is opened in response to depolarization of the cell membrane (for reviews see Hagiwara & Byerly, 1981, 1983; Tsien, 1983; Reuter, 1983). The Ca2+ flux creates a net inward, depolarizing current and the resulting accumulation of Ca2+ in the cytoplasm can act as a chemical trigger for secretion of hormones and neurotransmitters, contraction of muscle and a variety of other Ca2+-sensitive events. Thus, upon sensing membrane potential changes, Ca2+ channels simultaneously generate an electrical signal while directly creating an intracellular chemical messenger. This dual ability is unique among the family of ion channels and allows the Ca2+ channel to play a variety of roles in excitation-secretion and excitation-contraction coupling. It has now become clear that versatility of function is reflected by diversity of the types of Ca2+ channels on the membrane of individual cells. This article describes the nature of data which have demonstrated multiple channel types, reviews the literature suggesting that many cells have several kinds of Ca2+ channels, and discusses newer data regarding a neurotoxin that distinguishes among different Ca2+ channels