We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (∼45 pS at −100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.
Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes. Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+insensitivity has been used to infer that processes are not dependent on SACs. The limitations of this approach are not adequately appreciated, however. Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account. Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs. Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels. Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.
CHF promotes inducibility of sustained atrial tachycardia. Based on the mode of tachycardia induction, responses to pacing and calcium antagonists, and presence of DADs, atrial tachycardia in this CHF model has a mechanism most consistent with DAD-induced triggered activity resulting from intracellular calcium overload.
Characteristics and functions of the cardiac swelling-activated Cl current (I(Cl,swell)) are considered in physiologic and pathophysiologic settings. I(Cl,swell) is broadly distributed throughout the heart and is stimulated not only by osmotic and hydrostatic increases in cell volume, but also by agents that alter membrane tension and direct mechanical stretch. The current is outwardly rectifying, reverses between the plateau and resting potentials (E(m)), and is time-independent over the physiologic voltage range. Consequently, I(Cl,swell) shortens action potential duration, depolarizes E(m), and acts to decrease cell volume. Because it is activated by stimuli that also activate cation stretch-activated channels, I(Cl,swell) should be considered as a potential effector of mechanoelectrical feedback. I(Cl,swell) is activated in ischemic and non-ischemic dilated cardiomyopathies and perhaps during ischemia and reperfusion. I(Cl,swell) plays a role in arrhythmogenesis, myocardial injury, preconditioning, and apoptosis of myocytes. As a result, I(Cl,swell) potentially is a novel therapeutic target.
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