Of the four major types of Ca channel described in vertebrate cells (designated T. L, N, and P), N-type Ca channels are unique in that they are found spedflcafly in neurons, have been correlated with control ofneurotransmitter release, and are blocked by w-conotoxin, a neuropeptide toxin isolated from the marine snail Conus geographus. A set of overlapping cDNA clones were isolated and found to encode a Ca channel a-i subunit, desiated rbB-I. Polyclonal antiserum generated against a peptide from the rbB-I sequence selectively immunoprecipitates high-affinity 125I-labeled w-conotoxin-binding sites from labeled rat forebrain membranes. PCR analysis shows that, like N-type Ca channels, expression of rbB-I is limited to the nervous system and neuronally derived cell lines. This brain Ca channel may mediate the c-conotoxin-sensitive Ca influx required for neurotransmitter release at many synapses.Calcium entry into cells through voltage-gated Ca channels mediates a wide variety of cellular and physiological functions. Electrophysiological and pharmacological criteria have defined four main types of Ca channel in vertebrate cells (T, L, N, and P; for reviews see refs. 1 and 2). Of these, N-type Ca channels are distinct in that they have only been described in neurons and are blocked by the neuropeptide toxin, w-conotoxin GVIA (w-CgTx; refs. 1-7). The correlation between w-CgTx-sensitive N-type Ca channels and the sensitivity of neurotransmitter release to w-CgTx (8-11) has led to the proposal that N-type Ca channels are concentrated at presynaptic nerve termini and that they play a major role in mediating chemical synaptic transmission (12 EXPERIMENTAL PROCEDURES Isolation of rbB cDNAs. We previously isolated 10 partial rat brain cDNAs that were designated rbB-type Ca channels (32). To isolate further rbB cDNAs, oligonucleotides were synthesized against the 5' region of the rbB-10 sequence and used to screen 600,000 plaque-forming units ofa size-selected rat brain cDNA library (32). The complete DNA sequence of five overlapping rbB cDNAs was determined for this study: rbB-1214 [5267 base pairs (bp)], rbB-1274 (5620 bp), rbB-10 (4789 bp), rbB-8 (7463 bp), and rbB-79 (5079 bp).Polycdonal Antisera Production. New Zealand White rabbits were immunized with peptides: rbA-I (residues 865-881; ref. 37) KYPSSPERAPGREGPYGRE; rbB-I (residues 851-867) KYRHHRHRDRDKTSASTPA; and rbC-II (residues 821-838; ref. 38) KYTTKINMDDLQPSENEDKS to yield the antisera CNA 1, CNB 1, and CNC 1 as described (39). The amino terminal K and Y residues of each peptide are not part of the corresponding Ca channel sequence and were added for coupling and radiolabeling purposes.Radiolabeling Brain Membranes and Immu recipitation. For immunoprecipitation one rat forebrain was homogenized in 15 ml of 320 mM sucrose containing 0.2 mM phenylmethylsulfonyl fluoride, pepstatin A at 1 ,ug/ml, and leupeptin at 1 ,ug/ml at 4°C. The homogenate was spun for 2 min at 5000 rpm (Sorval SS34 rotor), and the supernatant was collected. For each test 40 ,ug of pro...
Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K+ free pipette solutions that alter the K+ equilibrium potential to non-physiological voltages. We show that an astrocyte’s associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte’s VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K+ and thereby maintains a sustained driving force for highly efficient K+ uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.
The inhibition of L- and T-type Ca2+ currents by Gd3+ was studied in the rat pituitary GH4C1 cell line. In whole cell patch recordings, Gd3+ at concentrations of 50 nM to 5 microM blocked Ca2+ current through L-type channels. Block was promoted by prolonged channel activation. With 4.5-s test pulses to + 10 mV, Gd3+ at concentrations as low as 200 nM produced near-complete block of L current. At higher Gd3+ concentrations (5 microM), complete block occurred with short test pulses and appeared to be independent of channel activation. Gd3+ also blocked current through low-threshold T channels in GH4C1 cells. Two other trivalent elements, La3+ and Y3+, blocked L-type Ca2+ channels in GH4C1 cells with potency similar to Gd3+. These results indicate that these trivalent cations are effective nonselective inhibitors of both low- and high-threshold Ca2+ channels in endocrine cells. In this regard, they are among the most potent inorganic Ca2+ antagonists yet discovered.
Bovine adrenal zona fasciculata (AZF) cells express a background K؉ channel (I AC ) that sets the resting potential and acts pivotally in ACTH-stimulated cortisol secretion. We have cloned a bTREK-1 (KCNK2) tandempore K ؉ channel cDNA from AZF cells with properties that identify it as the native I AC . The bTREK-1 cDNA is expressed robustly in AZF cells and includes transcripts of 4.9, 3.6, and 2.8 kb. In patch clamp recordings made from transiently transfected cells, bTREK-1 displayed distinctive properties of I AC in AZF cells. Specifically, bTREK-1 currents were outwardly rectifying with a large instantaneous and smaller time-dependent component. Similar to I AC , bTREK-1 increased spontaneously in amplitude over many minutes of whole cell recording and was blocked potently by Ca 2؉ antagonists including penfluridol and mibefradil and by 8-(4-chlorophenylthio)-cAMP. Unitary TREK-1 and I AC currents were nearly identical in amplitude. The native I AC current, in turn, displayed properties that together are specific to TREK-1 K ؉ channels. These include activation by intracellular acidification, enhancement by the neuroprotective agent riluzole, and outward rectification. bTREK-1 current differed from native K ؉ current only in its lack of ATP dependence. In contrast to I AC , the current density of bTREK-1 in human embryonic kidney-293 cells was not increased by raising pipette ATP from 0.1 to 5 mM. Further, the enhancement of I AC current in AZF cells by low pH and riluzole was facilitated by, and dependent on, ATP at millimolar concentrations in the pipette solution. Overall, these results establish the identity of I AC K ؉ channels, demonstrate the expression of bTREK-1 in a specific endocrine cell, identify potent new TREK-1 antagonists, and assign a pivotal role for these tandem-pore channels in the physiology of cortisol secretion. The activation of I AC by ATP indicates that native bTREK-1 channels may function as sensors that couple the metabolic state of the cell to membrane potential, perhaps through an associated ATP-binding protein.
SUMMARY1. The effects of the trivalent cations yttrium (Y3 ), lanthanum (La3"), cerium (Ce3+), neodymium (Nd3+), gadolinium (Gd3+), holmium (Ho3"), erbium (Er3"), ytterbium (Yb3+ ) and the divalent cation nickel (Ni2") on the T-type voltage gated calcium channel (VGCC) were characterized by the whole-cell patch clamp technique using rat and human thyroid C cell lines.
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