Time filter

Source Type

Wu W.,Nora Eccles Harrison Cardiovascular Research and Training Institute | Gardner A.,Nora Eccles Harrison Cardiovascular Research and Training Institute | Sanguinetti M.C.,University of Utah
Journal of Physiology | Year: 2014

At depolarized membrane potentials, the conductance of some voltage-gated K+ channels is reduced by C-type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C-type inactivation in hERG1 K+ channels is voltage-dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C-type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C-type inactivation were introduced into subunits that were combined with wild-type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two-microelectrode voltage clamp was used to measure the kinetics and steady-state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C-type inactivation of hERG1 channels involves a concerted, all-or-none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the specific mutation used to probe the gating process. © 2014 The Physiological Society. Source

Swietach P.,Oxford Genetics | Spitzer K.W.,Nora Eccles Harrison Cardiovascular Research and Training Institute | Vaughan-Jones R.D.,Oxford Genetics
Cardiovascular Research | Year: 2015

Aims Contraction of the heart is regulated by electrically evoked Ca2+ transients (CaTs). H+ ions, the end products of metabolism, modulate CaTs through direct interactions with Ca2+-handling proteins and via Na+-mediated coupling between acid-extruding proteins (e.g. Na+/H+ exchange, NHE1) and Na+/Ca2+ exchange. Restricted H+ diffusivity in cytoplasm predisposes pH-sensitive Ca2+ signalling to becoming non-uniform, but the involvement of readily diffusible intracellular Na+ ions may provide a means for combatting this. Methods and results CaTs were imaged in fluo3-loaded rat ventricular myocytes paced at 2 Hz. Cytoplasmic [Na+] ([Na+]i) was imaged using SBFI. Intracellular acidification by acetate exposure raised diastolic and systolic [Ca2+] (also observed with acid-loading by ammonium prepulse or CO2 exposure). The systolic [Ca2+] response correlated with a rise in [Na+]i and sarcoplasmic reticulum Ca2+ load, and was blocked by the NHE1 inhibitor cariporide (CO2/HCO3 --free media). Exposure of one half of a myocyte to acetate using dual microperfusion (CO2/HCO3 --free media) raised diastolic [Ca2+] locally in the acidified region. Systolic [Ca2+] and CaT amplitude increased more uniformly along the length of the cell, but only when NHE1 was functional. Cytoplasmic Na+ diffusivity (DNa) was measured in quiescent cells, with strophanthidin present to inhibit the Na+/K+ pump. With regional acetate exposure to activate a local NHE-driven Na+-influx, DNa was found to be sufficiently fast (680 μm2/s) for transmitting the pH-systolic Ca2+ interaction over long distances. Conclusions Na+ ions are rapidly diffusible messengers that expand the spatial scale of cytoplasmic pH-CaT interactions, helping to co-ordinate global Ca2+ signalling during conditions of intracellular pH non-uniformity. © 2014 The Author. Source

Swietach P.,Oxford Genetics | Leem C.-H.,University of Ulsan | Spitzer K.W.,Nora Eccles Harrison Cardiovascular Research and Training Institute | Vaughan-Jones R.D.,Oxford Genetics
Journal of Physiology | Year: 2014

Cellular processes are exquisitely sensitive to H+ and Ca2+ ions because of powerful ionic interactions with proteins. By regulating the spatial and temporal distribution of intracellular [Ca2+] and [H+], cells such as cardiac myocytes can exercise control over their biological function. A well-established paradigm in cellular physiology is that ion concentrations are regulated by specialized, membrane-embedded transporter proteins. Many of these couple the movement of two or more ionic species per transport cycle, thereby linking ion concentrations among neighbouring compartments. Here, we compare and contrast canonical membrane transport with a novel type of Ca2+-H+ coupling within cytoplasm, which produces uphill Ca2+ transport energized by spatial H+ ion gradients, and can result in the cytoplasmic compartmentalization of Ca2+ without requiring a partitioning membrane. The mechanism, demonstrated in mammalian myocytes, relies on diffusible cytoplasmic buffers, such as carnosine, homocarnosine and ATP, to which Ca2+ and H+ ions bind in an apparently competitive manner. These buffer molecules can actively recruit Ca2+ to acidic microdomains, in exchange for the movement of H+ ions. The resulting Ca2+ microdomains thus have the potential to regulate function locally. Spatial cytoplasmic Ca2+-H+ exchange (cCHX) acts like a 'pump' without a membrane and may be operational in many cell types. © 2014 The Physiological Society. Source

Isaacson B.M.,Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc | Stinstra J.G.,Numira Biosciences | Bloebaum R.D.,University of Utah | Pasquina P.F.,U.S. Army | And 3 more authors.
IEEE Transactions on Biomedical Engineering | Year: 2011

Although the survival rates of warfighters in recent conflicts are among the highest in military history, those who have sustained proximal limb amputations may present additional rehabilitation challenges. In some of these cases, traditional prosthetic limbs may not provide adequate function for service members returning to an active lifestyle. Osseointegration has emerged as an acknowledged treatment for those with limited residual limb length and those with skin issues associated with a socket together. Using this technology, direct skeletal attachment occurs between a transcutaneous osseointegrated implant (TOI) and the host bone, thereby eliminating the need for a socket. While reports from the first 100 patients with a TOI have been promising, some rehabilitation regimens require 12-18 months of restricted weight bearing to prevent overloading at the bone-implant interface. Electrically induced osseointegration has been proposed as an option for expediting periprosthetic fixation and preliminary studies have demonstrated the feasibility of adapting the TOI into a functional cathode. To assure safe and effective electric fields that are conducive for osseoinduction and osseointegration, we have developed multiscale modeling approaches to simulate the expected electric metrics at the bone -implant interface. We have used computed tomography scans and volume segmentation tools to create anatomically accurate models that clearly distinguish tissue parameters and serve as the basis for finite element analysis. This translational computational biological process has supported biomedical electrode design, implant placement, and experiments to date have demonstrated the clinical feasibility of electrically induced osseointegration. © 2011 IEEE. Source

Swenson D.J.,University of Utah | Swenson D.J.,Scientific Computing and Imaging Institute | Geneser S.E.,Scientific Computing and Imaging Institute | Stinstra J.G.,Scientific Computing and Imaging Institute | And 5 more authors.
Annals of Biomedical Engineering | Year: 2011

The electrocardiogram (ECG) is ubiquitously employed as a diagnostic and monitoring tool for patients experiencing cardiac distress and/or disease. It is widely known that changes in heart position resulting from, for example, posture of the patient (sitting, standing, lying) and respiration significantly affect the body-surface potentials; however, few studies have quantitatively and systematically evaluated the effects of heart displacement on the ECG. The goal of this study was to evaluate the impact of positional changes of the heart on the ECG in the specific clinical setting of myocardial ischemia. To carry out the necessary comprehensive sensitivity analysis, we applied a relatively novel and highly efficient statistical approach, the generalized polynomial chaos-stochastic collocation method, to a boundary element formulation of the electrocardiographic forward problem, and we drove these simulations with measured epicardial potentials from whole-heart experiments. Results of the analysis identified regions on the body-surface where the potentials were especially sensitive to realistic heart motion. The standard deviation (STD) of ST-segment voltage changes caused by the apex of a normal heart, swinging forward and backward or side-to-side was approximately 0.2 mV. Variations were even larger, 0.3 mV, for a heart exhibiting elevated ischemic potentials. These variations could be large enough to mask or to mimic signs of ischemia in the ECG. Our results suggest possible modifications to ECG protocols that could reduce the diagnostic error related to postural changes in patients possibly suffering from myocardial ischemia. © 2011 Biomedical Engineering Society. Source

Discover hidden collaborations