PubMed · 431710
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1979. First aid recommendations changed.. https://pubmed.ncbi.nlm.nih.gov/431710/
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Tandem pore domain K+ channels represent a new family of ion channels involved in the control of background membrane conductances. We report the structural and functional properties of a TWIK-related acid-sensitive K+ channel (rTASK), a new member of this family cloned from rat cerebellum. The salient features of the primary amino acid sequence include four putative transmembrane domains and, unlike other cloned tandem pore domain channels, a PDZ (postsynaptic density protein, disk-large, zo-1) binding sequence at the C terminal. rTASK has distant overall homology to a putative Caenorhabditis elegans K+ channel and to the mammalian clones TREK-1 and TWIK-1. rTASK expression is most abundant in rat heart, lung, and brain. When exogenously expressed in Xenopus oocytes, rTASK currents activate instantaneously, are noninactivating, and are not gated by voltage. Because rTASK currents satisfy the Goldman-Hodgkin-Katz current equation for an open channel, rTASK can be classified an open rectifier. Activation of protein kinase A produces inhibition of rTASK, whereas activation of protein kinase C has no effect. rTASK currents were inhibited by extracellular acidity. rTASK currents also were inhibited by Zn2+ (IC50 = 175 microM), the local anesthetic bupivacaine (IC50 = 68 microM), and the anti-convulsant phenytoin ( approximately 50% inhibition at 200 microM). By demonstrating open rectification and open probability independent of voltage, we have established that rTASK is a baseline potassium channel.
Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.
The effects of long-term exposure to hyperosmotic medium on the Na+/H+ exchanger isoform NHE-3 were examined in cultured renal epithelial cells (LLC-PK1). LLC-PK1 cells were grown to confluence in control medium (310 mOsm/kg H2O) and then either switched to a hyperosmotic medium (510 mOsm/kg H2O; addition of NaCl or mannitol) or maintained in the control medium for 48 hours. The Na+/H+ exchanger activity was then assessed in isosmotic solutions by measurement of amiloride-sensitive acid-stimulated 22Na+ influx or Na+-dependent acid extrusion. Acid-stimulated 22Na+ influx was decreased significantly in cells incubated in hyperosmotic medium (10.5 +/- 0.9 nmol/mg protein, control vs. 5.8 +/- 0.6, hyperosmotic; P < 0.01). Incubation in hyperosmotic medium also decreased the initial rate of Na+-dependent acid extrusion by approximately 60% over the intracellular pH range 6.9 to 7.3. Intracellular buffering power did not differ in the control and hyperosmotic groups. The Na+/H+ exchanger isoform NHE-3 mRNA and protein, assessed by Northern hybridization and immunoblot analysis, respectively, were unchanged in LLC-PK1 cells incubated in hyperosmotic medium compared with controls, suggesting post-translational regulation by high osmolality. These results demonstrate that long-term exposure to hyperosmotic medium causes an adaptive decrease in Na+/H+ exchange (NHE-3) activity in LLC-PK1 cells, and that this effect is unlikely to involve antiporter gene regulation or a change in protein abundance.