The fast-inactivation process in the hERG channel can be affected by mutations in the pore or S6 domain, similar to the C-type inactivation in the Shaker channel. However, differences in the kinetics and voltage dependence of inactivation between these two channels suggest that different structural determinants may be involved. To explore this possibility, we mutated a serine in the outer mouth region of hERG (S631) to residues of different physicochemical properties and compared the resulting changes in the channel's inactivation process with those resulting from mutations of an equivalent position in the Shaker channel (T449). The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker. S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker. To further study why there are such differences, we replaced S631 with cysteine. This allowed us to manipulate the properties of thiol groups at position 631 and correlate side-chain properties here with changes in channel function. S631C behaved like the wild-type channel when the thiol groups were in the reduced state. Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E. The same thiol-modifying maneuvers did not affect the wild-type channel function. Our results suggest differences in the outer mouth structure between hERG and Shaker, and we propose a "molecular spring" hypothesis to explain these differences.
,B-Adrenergic agonists are known to potentiate the force of cardiac contraction and accelerate the rate of its relaxation. The increase in the force of contraction results primarily from enhanced Ca2+ current and Ca2+ release secondary to cAMPdependent phosphorylation of the Ca2+ channel (1-4). On the other hand, the phosphorylation of phospholamban and the subsequent stimulation of Ca2+ pump (5-7), in addition to decreased myofilament Ca2+ sensitivity (8-10), are thought to mediate the relaxant properties of 13-agonists. Quite similar to the mammalian myocardium, the frog heart also exhibits some of the characteristic features associated with the (3-agonist response, namely potentiation of phasic contraction (twitch) followed by enhanced inhibition of maintained (tonic) tension (11)(12)(13)(14). In light of recent reports suggesting low content of Ca-ATPase (15, 16), absence of mRNA message for CaATPase (17), and lack of functionally significant Ca2+-release stores (18)(19)(20)(21)(22) in the frog heart, the (3-agonist tensionsuppressant effect is surprising and unexpected if its similarity to the mammalian heart is solely mediated by the cAMPinduced phosphorylation of the phospholamban. The possibility that the Na+-Ca2+ exchanger significantly contributes to the sequestration of Ca2+ in the frog heart was suggested from early findings that (i) developed tension was dominated by a tonic component with sigmoid voltage dependence (19) similar to that described for Na+-Ca2+ exchanger current, INa-Ca; (ii) the rate of relaxation of contraction in the frog myocardium was markedly suppressed (from 100 ms to 4.5 s) when Na+ was omitted from the extracellular solutions; and (iii) catacholamines failed to accelerate the rate of relaxation of contraction or suppress tonic KCl-induced contractures (14).To examine the possible involvement of Na+-Ca2+ exchanger in mediating the (3-agonist tension-suppressant (relaxant) effect in the frog ventricular myocardium, we studied the effect of f3-agonists on INaCa in isolated frog ventricular myocytes.Our experiments revealed a novel regulatory property of isoproterenol mediated via the 13-receptor/adenylate-cyclase/ cAMP-dependent cascade. This mechanism, which results in a decreased Ca2+1oad on the cardiac myocytes during experimentally or hormonally induced prolonged membrane depolarizations, may be responsible for catacholamine-induced suppression of KCl-induced contractures (11,13,14) and uncoupling of the duration of contraction and the action potential (11,13,14), resulting in enhanced relaxation of the twitch.MATERIALS AND METHODS Cell Isolation, Experimental Protocol, and Data Analysis. Frog (Rana pipiens) ventricular myocytes were enzymatically isolated (23) and whole-cell clamped using 2-to 5-Mfl patch pipettes (24). A patch clamp amplifier (Dagan Instruments, Minneapolis; model 9000) was used to voltage-clamp isolated myocytes. The data were collected, stored, and analyzed on a personal computer using PCLAMP 5.51 (Axon Instruments, Foster City, CA) and ORIGIN (Microcal,...
Despite the widespread use of traditional Chinese medicine (TCM) in clinical settings, proving its effectiveness via scientific trials is still a challenge. TCM views the human body as a complex dynamical system, and focuses on the balance of the human body, both internally and with its external environment. Such fundamental concepts require investigations using system-level quantification approaches, which are beyond conventional reductionism. Only methods that quantify dynamical complexity can bring new insights into the evaluation of TCM. In a previous article, we briefly introduced the potential value of Multiscale Entropy (MSE) analysis in TCM. This article aims to explain the existing challenges in TCM quantification, to introduce the consistency of dynamical complexity theories and TCM theories, and to inspire future system-level research on health and disease.
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