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E W McCleskey

Publications and source records attributed to E W McCleskey.

49 records · Page 3Linked to original sources
PubMed1990

Two components of high-threshold Ca2+ current inactivate by different mechanisms.

High-threshold Ca2+ current triggers neurotransmitter release, but the existence, significance, and correct identification of different types of high-threshold Ca2+ channels remain controversial. We show selective inhibition of a rapidly inactivating component of high-threshold Ca2+ current in rat sensory neurons by bursts of brief pulses that mimic trains of action potentials and by prolonged depolarization just above the normal rest potential. In contrast, a slowly inactivating component decreases only when sufficient Ca2+ accumulates within the cell. Thus, there are physiologically important differences: whereas availability of the transient component depends on the value of the rest potential and the pattern of a prior stimulus, the sustained component seems to provide a baseline level of voltage-dependent Ca2+ entry that is lost only when intracellular Ca2+ rises.

Animals↗
PubMed1990

T-type calcium channels: heterogeneous expression in rat sensory neurons and selective modulation by phorbol esters.

We report selective inhibition of low-threshold, T-type calcium channels by a phorbol ester in rat sensory neurons. Cells were exposed, either acutely or with 15-60 min preincubations, to low concentrations of phorbol 12-myristate 13-acetate, (PMA), an activator of protein kinase C; if the temperature was 29 degrees C or higher, T-type Ca current was diminished without effect on high-threshold Ca current. In untreated cells, the amplitude of T-type Ca current varies widely among neighboring sensory neurons. T channels are absent in about 25% of cells, provide a small current near threshold for the majority of cells, and are a dominant pathway for calcium entry in a small subset of neurons. The results are of interest because, by selectively inhibiting a calcium channel expressed differently among subpopulations of sensory neurons, activation of protein kinase C might selectively suppress particular sensations.

Animals↗
PubMed1988

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.

Animals↗
PubMed1987

Omega-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle.

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.

Animals↗
PubMed1986

Different types of calcium channels.

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

The Ca channel in skeletal muscle is a large pore.

The permeability of Ca channels to various foreign cations has been investigated in the absence of external Ca2+. All physiological metal cations are clearly permeant, including Mg2+. The large organic cation n-butylamine+ is sparingly permeant or impermeant, but its larger derivative 1,4-diaminobutane2+ is highly permeant. Among the cations of the methylated ammonium series, permeability diminishes in a graded fashion as ion size increases. Tetramethylammonium, the largest cation found to be permeant, has a diameter of about 6 A; hence, the aqueous pore of the Ca channel at its narrowest point can be no smaller. That the pore is so large strengthens our view that, under physiologic conditions, the high selectivity of Ca channels is due to selective binding of Ca2+ rather than to rejection of other cations by, for example, a sieving mechanism.

Animals↗
PubMed1985

Calcium channels and intracellular calcium release are pharmacologically different in frog skeletal muscle.

The pharmacology of Ca2+ channels and intracellular Ca2+ release from the sarcoplasmic reticulum (s.r.) were compared by injecting Ca2+ channel blockers into the cytoplasm and observing contraction under voltage clamp of frog skeletal muscle fibres, a preparation that contracts only in response to Ca2+ release from the s.r. A method for quantifying intracellular injections by co-injecting a fluorescent dye is described. Nifedipine injected into cells blocks Ca2+ current through the cell membrane showing that nifedipine is active when applied to the cytoplasmic side of the membrane in which Ca2+ channels are located. Neither the presence of Ca2+ channel blockers in the extracellular medium nor 24 h incubation in nifedipine and D-600 affect contraction. Nifedipine and D-600 injected to intracellular concentrations much greater than necessary to block Ca2+ channels do not affect contraction. The presence of 30 microM-D-600 during K+ contractures caused paralysis but 20 microM-nifedipine did not. Thus, contracture-dependent D-600 paralysis is not due to blockade of the transverse tubule Ca2+ channel. It is concluded that: (a) a functioning Ca2+ channel on the cell membrane is not necessary to trigger Ca2+ release from the s.r.; (b) s.r. Ca2+ release and Ca2+ channels are pharmacologically different.

Action Potentials↗
PubMed1984

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.

Action Potentials↗
PubMed1984

Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore.

Voltage-clamp studies were carried out to compare currents through Ca2+ channels (ICa) with Na+ currents (Ins) through a non-selective cation conductance blocked by micromolar concentrations of external Ca2+. The gating of both currents was found to have similar time and voltage dependence. The amplitudes of ICa and Ins varied widely, but Ins was always large in fibres with large ICa, and small in fibres with small ICa. Both ICa and Ins were blocked by the specific Ca2+ channel blocker nifedipine, with half-blockage concentrations that were virtually identical (KD = 0.9 microM for ICa and 0.7 microM for Ins). ICa and Ins were also equally sensitive to block by diltiazem (KD = 80 microM). These parallels between Ins and ICa are most easily explained if Ins flows through Ca2+ channels. Apparently, Ca2+ channels bear high-affinity Ca2+-binding sites, and are highly permeable to monovalent cations when Ca2+ is absent. Ba2+ currents (IBa) and ICa were measured in external solutions containing mixtures of Ba2+ and Ca2+. IBa is blocked by Ca2+, as is Ins. Adding Ba2+ to Ca2+ produces only small or no increases in current, as if Ba2+ is only sparingly permeant when Ca2+ is present. Membrane currents in Ba2+/Ca2+ mixtures show anomalous mole-fraction behaviour, suggesting that Ca2+ channels are single-file, multi-ion pores. Complex current transients are observed under maintained depolarizations in Na+/Ca2+ and Ba2+/Ca2+ mixtures. They suggest that in ion mixtures, Ca2+ channels transport Ca2+ in preference to Na+ and Ba2+. Hence Ca2+ channels are selective for Ca2+, even though current amplitudes suggest that the Na+ or Ba2+ permeabilities in the absence of Ca2+ are as high as, or higher than, the Ca2+ permeability. We conclude that the selective permeability of Ca2+ channels depends on the presence of Ca2+. In model calculations, our observations are explained as a consequence of Ca2+ channels being single-file pores. It is proposed that Ca2+ channels derive much of their ion selectivity from high-affinity Ca2+ binding sites located in an otherwise unselective aqueous pore.

Action Potentials↗
PubMedDate not supplied

Dihydropyridine receptors in muscle are voltage-dependent but most are not functional calcium channels.

1,4-Dihydropyridines are a new class of compounds believed to bind specifically and with high affinity to voltage-dependent calcium channels. They may be the first example of a ligand of use in the extraction and purification of the Ca channel. Although Ca channels and dihydropyridine receptors are found in many tissues, the richest and most convenient source is skeletal muscle. Functionally, 1,4-dihydropyridines such as nifedipine and nitrendipine block Ca channels; this effect is believed to form the basis for their clinical importance as Ca antagonists in relaxing vascular smooth muscle. But where currents through Ca channels can be measured directly, the block has required 100-1,000 times higher concentrations of dihydropyridine than necessary for the saturation of dihydropyridine binding sites. This discrepancy has remained unresolved because the study of pharmacological effects on Ca channels has required intact cells, while it has been difficult to investigate binding in other than cell-free preparations. Here we describe a method for measuring dihydropyridine binding to intact skeletal muscle and we compare our results with voltage-clamp measurements of Ca-channel block. We conclude that less than a few per cent of the binding sites in skeletal muscle represent functional Ca channels, contrary to general belief.

Animals↗