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D E Clapham

Publications and source records attributed to D E Clapham.

At least 91 records · Page 5Linked to original sources
PubMed1994

Hypotonicity activates a native chloride current in Xenopus oocytes.

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G-protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium-activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.

Animals↗
PubMed1993

New roles for G-protein beta gamma-dimers in transmembrane signalling.

When a membrane-bound receptor acts on a G protein, the GTP-binding or G alpha subunit dissociates from the G beta gamma dimer. Until recently, the G alpha subunit alone was thought to act on the enzymes and ion channels controlled by these proteins. Newer evidence indicates that the G beta gamma dimer also plays a major part in signal transmission, enhancing the complexity of the possible interactions between the G proteins and their targets.

Animals↗
PubMed1992

Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes.

Following receptor activation in Xenopus oocytes, spiral waves of intracellular Ca2+ release were observed. We have identified key molecular elements in the pathway that give rise to Ca2+ excitability. The patterns of Ca2+ release produced by GTP-gamma-S and by inositol 1,4,5-trisphosphate (IP3) are indistinguishable from receptor-induced Ca2+ patterns. The regenerative Ca2+ activity is critically dependent on the presence of IP3 and on the concentration of intracellular Ca2+, but is independent of extracellular Ca2+. Broad regions of the intracellular milieu can be synchronously excited to initiate Ca2+ waves and produce pulsating foci of Ca2+ release. By testing the temperature dependence of wavefront propagation, we provide evidence for an underlying process limited by diffusion, consistent with the elementary theory of excitable media. We propose a model for intracellular Ca2+ signaling in which wave propagation is controlled by IP3-mediated Ca2+ release from internal stores, but is modulated by the cytoplasmic concentration and diffusion of Ca2+.

Animals↗
PubMed1992

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.

Adenosine Triphosphate↗
PubMed1992

Spiral waves and intracellular calcium signalling.

Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intracellular Ca2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular Ca2+ release in Xenopus oocytes, introduce the concept of Ca2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information.

Animals↗
PubMed1991

Cloning and expression of a rat cardiac delayed rectifier potassium channel.

We have cloned a cDNA (designated RAK) coding for a delayed-rectifier K current (IRAK) from adult rat heart atrium and expressed it in Xenopus oocytes. RAK differs from the cloned rat brain K current, BK2 [McKinnon, D. (1989) J. Biol. Chem. 264, 8230-8236], by one amino acid at residue 411. RAK expressed in oocytes compares closely to the intrinsic adult rat atrial delayed-rectifier current measured by using whole-cell recording of single isolated cells. Northern blot analysis confirmed the presence of the channel in adult rat atrium, and to a lesser extent, in rat ventricle. IRAK activates with time constants ranging from 58 ms at -20 mV to 6 ms at +60 mV and does not show significant inactivation over 800 ms. It is blocked by 4-aminopyridine greater than barium much greater than tetraethylammonium chloride, which is similar to the relative potencies of these blockers on the native delayed rectifier current. We conclude that the main delayed rectifier K current in adult rat atria is virtually identical to a neuronal delayed rectifier, BK2.

4-Aminopyridine↗
PubMed1991

Chloride channels in the nuclear membrane.

Chloride-selective ion channels were measured from isolated rat liver nuclei. Single ion channel currents were recorded in both "nuclear-attached" and in excised patches in the inside-out configuration of the patch-clamp technique. Two types of chloride conductance were defined, a large conductance (150 pS; iCl,N) channel with complex kinetics and multiple substates, and a second smaller conductance (58 pS;ICln) channel sensitive to block by ATP. The channels were inhibited by pharmacological agents known to block chloride channels and were insensitive to internal and external changes in calcium and magnesium. Presumably the channels reside in the external membrane of the nuclear double membrane and may mediate charge balance in the release and uptake of calcium from the perinuclear space.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Two novel cardiac atrial K+ channels, IK.AA and IK.PC.

Two K(+)-selective channels in neonatal rat atrial cells activated by lipophilic compounds have been characterized in detail. The arachidonic acid-stimulated channel (IK.AA) had a slope conductance of 124 +/- 17 pS at +30 mV in symmetrical 140 mM potassium and a mean open time of approximately 1 ms, and was relatively voltage independent. IK.AA activity was reversibly increased by lowering pH to 6.0. Arachidonic acid was most effective in activating this channel, although a number of lipophilic compounds resulted in activation. Surprisingly, choline, a polar molecule, also activated the channel. A second K+ channel was activated by 10 microM phosphatidylcholine applied to the intracellular surface of inside-out atrial patches. This channel (IK.PC) had a slope conductance of 60 +/- 6 pS at +40 mV and a mean open time of approximately 0.6 ms, and was also relatively voltage independent. Fatty acids are probably monomeric in the membrane under the conditions of our recording; thus detergent effects are unlikely. Since a number of compounds including fatty acids and prostaglandins activated these two channels, an indirect, channel-specific mechanism may account for activation of these two cardiac K+ channels.

Animals↗
PubMed1990

Intracellular regulation of ion channels in cell membranes.

Cells communicate with their environment through receptor proteins on the cell membrane. Some ion channels are receptors, whereas others are linked to receptors through guanine nucleotide-binding proteins (G proteins). Ion channels control intracellular concentrations of ions such as calcium, and these concentrations control cell functions such as secretion and cell division. This review summarizes the current state of knowledge about the control of ion channels.

Cell Communication↗
PubMed1989

Potassium channels in cardiac cells activated by arachidonic acid and phospholipids.

Two types of potassium-selective channels activated by intracellular arachidonic acid or phosphatidylcholine have been found in neonatal rat atrial cells. In inside-out patches, arachidonic acid and phosphatidylcholine each opened outwardly rectifying potassium-selective channels with conductances of 160 picosiemens (IK.AA) and 68 picosiemens (IK.PC), respectively. These potassium channels were not sensitive to internally applied adenosine triphosphate (ATP), magnesium, or calcium. Lowering the intracellular pH from 7.2 to 6.8 or 6.4 reversibly increased IK.AA channel activity three- or tenfold, respectively. A number of fatty acid derivatives were tested for their ability to activate IK.AA. These potassium-selective channels may help explain the increase in potassium conductance observed in ischemic cells and raise the possibility that fatty acid derivatives act as second messengers.

Animals↗