K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+channels. The aim of this study was to examine the types of K+ channels involved in the contractility of longitudinal smooth muscle of rabbit small intestinein vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions (AU)

La mayoría de las células excitables, incluyendo las células lisas gastrointestinales, expresan varios tipos de canales de K+. El objetivo de este estudio es examinar los tipos de canales de K+que están involucrados en la contractilidad del músculo liso longitudinal del intestino delgado de conejoin vitro. Las contracciones espontáneas y las producidas por KCl se redujeron por atropina, fentolamina, propranolol, suramina, tetrodotoxina e indometacina. La amplitud y tono de las contracciones espontáneas aumentaron por apamin, charybdotoxina, iberiotoxina, E4031, tetraetilamonio (TEA) y BaCl2, mientras que la frecuencia de las contracciones se redujo en presencia de apamin, charybdotoxina y TEA. La 4-aminopiridina aumentó el tono de las contracciones espontáneas y redujo la amplitud y frecuencia de las contracciones. La glibenclamida no modificó la amplitud, frecuencia y tono de las contracciones. Las contracciones producidas por el KCl aumentaron en presencia de E4031, no fueron modificadas por el apamin, glibenclamida, NS1619 o diazóxida y disminuyeron en presencia de la charybdotoxina, TEA, 4-aminopiridina o BaCl2. Estos resultados sugieren que los canales de K+ activados por Ca2+ de pequeña y gran conductancia, canales de K+ HERG canales de K+ rectificadores de entrada participan en las contracciones espontáneas del intestino delgado. Por otra parte, los canales de K+ voltaje-dependientes, canales de K+ HERG, canales de K+ rectificadores de entrada y canales de K+activados por Ca2+ de gran conductancia están implicados en las contracciones producidas por el KCl (AU)

Role of K(+) channels in the coronary and renal vascular reactivity to vasopressin in diabetic rats.

To study the role of K(+) channels in the coronary and renal vascular response to vasopressin during diabetes mellitus, and whether there are gender differences in this role, we have examined the isometric response to this peptide of 2-mm-long arterial segments from male and female, normoglycemic and streptozotocin-induced diabetic rats. Vasopressin (10(-12)-3 x 10(-8) M) produced arterial concentration-dependent contraction, and during normoglycemia, this contraction was lower in coronary arteries from female than from male rats, and it was similar in renal arteries from both genders. This contraction was reduced by diabetes in coronary arteries, and increased in renal arteries, from both genders. The blocker of Ca(2+)-sensitive K(+) channels charybdotoxin (10(-7) M) increased the contraction to vasopressin in coronary arteries of diabetic females, but not in the other cases (diabetic males and normoglycemic females or males). This blocker also increased the contraction to vasopressin in renal arteries from diabetic, but not in those from normoglycemic female rats, and also increased it in a higher magnitude in arteries from diabetic than in those from normoglycemic male rats. The blocker of ATP-sensitive K(+) channels glybenclamide (10(-5) M) or the scavenger of superoxide radicals superoxide dismutase (100 U/ml) did not modify the contraction to vasopressin in any experimental group. These results suggest that diabetes activates the modulatory role of K(+) channels in the coronary and renal vasoconstriction to vasopressin, but it alters in a different way the vasoconstriction to vasopressin in these two types of arteries. The effects of diabetes on this vasoconstriction are not related to increased release of superoxide radicals.

Multiple binding sites for melatonin on Kv1.3.

Melatonin is a small amino acid derivative hormone of the pineal gland. Melatonin quickly and reversibly blocked Kv1.3 channels, the predominant voltage-gated potassium channel in human T-lymphocytes, acting from the extracellular side. The block did not show state or voltage dependence and was associated with an increased inactivation rate of the current. A half-blocking concentration of 1.5 mM was obtained from the reduction of the peak current. We explored several models to describe the stoichiometry of melatonin-Kv1.3 interaction considering one or four independent binding sites per channel. The model in which the occupancy of one of four binding sites by melatonin is sufficient to block the channels gives the best fit to the dose-response relationship, although all four binding sites can be occupied by the drug. The dissociation constant for the individual binding sites is 8.11 mM. Parallel application of charybdotoxin and melatonin showed that both compounds can simultaneously bind to the channels, thereby localizing the melatonin binding site out of the pore region. However, binding of tetraethylammonium to its receptor decreases the melatonin affinity, and vice versa. Thus, the occupancy of the two separate receptor sites allosterically modulates each other.

A point mutation in the maxi-K clone dSlo forms a high affinity site for charybdotoxin.

This work investigated the interaction of CTX with two cloned analogues of the maxi-K channel, dSlo and hSlo. dSlo has been reported to be CTX insensitive. Single channel analysis revealed that dSlo was weakly blocked by the toxin, with a very high K(D) of 5.8 microM. The hSlo channels bound wild-type, recombinant CTX with high affinity and in a bimolecular fashion, and displayed a half-blocking concentration (K(D)) of 36 nM. A glutamate residue was substituted for the wild-type threonine at position 290 in dSlo. The mutant channel was expressed in COS-7 cells and reconstituted into lipid bilayers for single channel analysis. The mutant channel bound wild-type, recombinant CTX with high affinity and in a bimolecular fashion, and displayed a half-blocking concentration (K(D)) of 23 nM. Changing just one residue from threonine to glutamate at position 290 in dSlo changed the affinity of the channel's CTX-receptor over 100-fold. Kinetic analysis revealed that this large increase in affinity was due to decreasing the dissociation rate of the toxin. These results suggest that a CTX receptor does exist in the dSlo channel mouth and that the threonine at position 290 destabilizes the toxin on the binding site.