Conductivity and geometrical factors affecting volume measurements with an impedancimetric catheter

With a model based on Poisson’s equation, computations employing published data and measurements made in glass cylinders filled with saline, the influence of conductivity and geometry on the determination of volume was analysed with an impedancimetric catheter. Two distal electrodes inject a constant current while a set of central electrodes sense by sections the potential along the cylindrical chamber. It was concluded that each section ought to be independently calibrated, the distribution of the electrodes along the catheter and its relationship to the chamber radius being of paramount importance for a linear calibration. The ratio σ _r , of the external medium conductivity σ _e to the internal conductivity σ _i influences drastically both the linearity and the calibration of each section. If a section volume is linearly related to the total chamber volume, that section admittance is a total volume estimator.     

Real-time extraction of tissue impedance model parameters for electrical impedance spectrometer

This paper presents a new algorithm for real-time extraction of tissue electrical impedance model parameters from in vivo electrical impedance spectroscopic measurements. This algorithm was developed as a part of a system for muscle tissue ischemia measurements using electrical impedance spectroscopy. An iterative least square fitting method, biased with a priori knowledge of the impedance model was developed. It simultaneously uses both the real and imaginary impedance spectra to calculate tissue parameters R₀, R_∞, α and τ. The algorithm was tested with simulated data, and during real-time in vivo ischemia experiments. Experimental results were achieved with standard deviations of , σ_α=0.72%, and σ_τ=1.26%. On a Pentium II based PC, the algorithm converges to within 0.1% of the results in 17 ms. The results show that the algorithm possesses excellent parameter extraction capabilities, repeatability, speed and noise rejection.     

Effect of intracellular anisotropy on electrical source determination in a muscle fibre

Expressions are available for describing, quantitatively, the source associated with an action potential propagating along an excitatble fibre. For a nerve fibre one such expression defines an equivalent volume dipole density function τ(x) = − ∂/∂x (σ_i φ_i(x) − σ_e φ_e(x))a_x (where x is the axial co-ordinate, i is the intracellular and e the extracellular region, σ_i and σ_e are isotropic conductivities, ϕ the potential at the membrane, while axial symmetry is assumed), and this source fills the intracellular region. This source, as distinct from transmembrane current formulations, lies in a uniform, isotropic, extracellular, medium. Consequently, for a fibre bundle a simple superposition of sources, all lying in a uniform, isotropic, extracellular space, can be accomplished. However, for muscle fibres the presence of non-conducting myofibrils causes the intracellular space to be anisotropic. The paper describes the modification in the aforementioned expressions for the case of longitudinal and transverse propagation and extrapolation to an arbitrary angle of propagation. The resultant source continues to be expressed relative to a uniform, isotropic, extracellular medium.     

Electrical Conductivities of the Freshly Excised Cerebral Cortex in Epilepsy Surgery Patients; Correlation with Pathology, Seizure Duration, and Diffusion Tensor Imaging

Summary The electrical conductivities (σ) of freshly excised neocortex and subcortical white matter were studied in the frequency range of physiological relevance for EEG (5–1005 Hz) in 21 patients (ages 0.67 to 55 years) undergoing epilepsy neurosurgery. Surgical patients were classified as having cortical dysplasia (CD) or non-CD pathologies. Diffusion tensor imaging (DTI) for apparent diffusion coefficient (ADC) and fractional anisotropy (FA) was obtained in 9 patients. Results found that electrical conductivities in freshly excised neocortex vary significantly from patient to patient (σ = 0.0660–0.156 S/m). Cerebral cortex from CD patients had increased conductivities compared with non-CD cases. In addition, longer seizure durations positively correlated with conductivities for CD tissue, while they negatively correlated for non-CD tissue. DTI ADC eigenvalues inversely correlated with electrical conductivity in CD and non-CD tissue. These results in a small initial cohort indicate that electrical conductivity of freshly excised neocortex from epilepsy surgery patients varies as a consequence of clinical variables, such as underlying pathology and seizure duration, and inversely correlates with DTI ADC values. Understanding how disease affects cortical electrical conductivity and ways to non-invasively measure it, perhaps through DTI, could enhance the ability to localize EEG dipoles and other relevant information in the treatment of epilepsy surgery patients.     

Ex vivo biomechanical behavior of abdominal aortic aneurysm: Assessment using a new mathematical model

Knowledge of the biomechanical behavior of abdominal aortic aneurysm (AAA) as compared to nonaneurysmal aorta may provide information on the natural history of this disease. We have performed uniaxial tensile testing of excised human aneurysmal and nonaneurysmal abdominal aortic specimens. A new mathematical model that conforms to the fibrous structure of the vascular tissue was used to quantify the measured elastic response. We determined for each specimen the yield σ_y and ultimate σ_u strengths, the separate contribution to total tissue stiffness by elastin (E _E) and collagen (E _C) fibers, and a collagen recruitment parameter (A), which is a measure of the tortuosity of the collagen fibers. There was no significant difference in any of these mechanical properties between longitudinal and circumferential AAA specimens, nor inE _E andE _C between longitudinally oriented aneurysmal and normal specimens.A, σ_y, and σ_u were all significantly higher for the normal than for the aneurysmal group:A=0.223±0.046versus A=0.091±0.009 (mean ± SEM;p<0.0005), σ_y versus σ_y (p<0.05), and σ_u versus σ_u (p<0.0005), respectively. Our findings suggest that the AAA tissue is isotropic with respect to these mechanical properties. The observed difference inA between aneurysmal and normal aorta may be due to the complete recruitment and loading of collagen fibers at lower extensions in the former. Our data indicate that AAA rupture may be related to a reduction in tensile strength and that the biomechanical properties of AAA should be considered in assessing the severity of an individual aneurysm.     

Control of myotube contraction using electrical pulse stimulation for bio-actuator

The contractility of tissue-engineered muscle on the application of electrical signals is required for the development of bio-actuators and for muscle tissue regeneration. Investigations have already reported on the contraction of myotubes differentiated from myoblasts and the construction of tissue-engineered skeletal muscle using electrical pulses. However, the relationship between myotube contraction and electrical pulses has not been quantitatively evaluated. We quantitatively investigated the effect of electrical pulse frequency on the excitability of myotubes and developed bio-actuators made of tissue-engineered skeletal muscle. C2C12 cells were seeded on a collagen-coated dish and in collagen gel and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and antibiotics. When the cells reached confluence or after 2 days in culture, the medium was shifted to DMEM containing 7% horse serum to allow them to differentiate to C2C12 myotubes. We electrically stimulated the myotubes and tissue-engineered skeletal muscle, and contractions were observed under a microscope. The myotubes contracted synchronously with electrical pulses between 0.5 and 5 Hz and unfused tetanus was generated at 10 Hz. The contractile performance of tissue-engineered skeletal muscle made of collagen gel and C2C12 was similar to that of the myotubes. Both the rheobase and chronaxie of the myotubes were lowest when the electric field was applied parallel to the myotube axis, and the values were 8.33 ± 2.78 mA and 1.19 ± 0.38 ms, respectively. The motion of C2C12 myotube contraction depended on the pulse frequency and showed anisotropy in the electric field. These results suggest that a tissue-engineered bio-actuator may be controlled using electrical signals.     

Expression of sulphonylurea receptors in bovine monocytes from animals with a different metabolic rate

 The aim of this study was to investigate the relationship between expression of sulphonylurea receptors (SUR) and metabolic rate (MR). SUR on monocytes and cells from muscle tissue were detected using fluorescent glibenclamide and flow cytometry. Transmembrane potential differences were detected by oxonol dye fluorescence measurements. A bovine model was used to induce differences in the MR by exposure to different ambient temperatures (4°C and 18°C), by different feeding levels (1.0- and 1.6-fold the metabolizable energy requirement for maintenance) and by α₂-adrenergic stimulation. We found that cells from skeletal muscle (m. semimembranosus), immunochemically identified as smooth muscle cells, skeletal muscle fibres and monocytes, responded in comparable fashions to glibenclamide and ATP, i.e. with a depolarization, and to cromakalim with a polarization, suggesting that monocytes are useful indicators of regulatory events occurring in muscle cells. Glibenclamide fluorescence was assumed to represent SUR associated with K_ATP channels. Significant differences were detected in the percentage of depolarized monocytes in the different variants of the model. A linear correlation between monocytes that bound fluorescent glibenclamide and the MR was evident (with a coefficient of determination of 0.94) and was reproducible following reduction of the MR, by α₂-adrenergic stimulation, suggesting that expression is involved in the regulation of whole-body energy expenditure. Received: 4 February 1997 / Received after revision: 28 April 1997 / Accepted: 13 June 1997     

Impedance of a goat eye lens

The complex electrical impedance of a goat eye lens is studied in the frequency range 10mHz−10Hz at room temperature, using a computer-controlled AC impedance system. AC impedance software (model 368, version 2.2) is employed to determine the total impedance and capacitance of the eye lens at various frequencies. A Cole-Cole plot of the eye lens material is drawn between the real component of impedance Z′ and the imaginary component Z″ for each excitation frequency that shows a perfect arc of a semicircle, with its centre lying below the abscissa at an angle of 35°. The half-angle ϕ between R₀ and R_∞ is found to be 55°, which mathematically demonstrates the selective permeability of the eye lens. Using graphical analysis of the Cole-Cole plot, characteristic frequency f_c and distribution factor α are observed to be 1Hz and 0.77, respectively. At characteristic frequency, capacitance and total impedance are found to be 1.14μF and 9.08kΩ. The effect of electrode polarisation on capacitance is corrected, based on Fricke's power function. The observed electrical parameters are then used to explain the multiple current path through various tissue compartments. Further, an attempt is made to explain the results on the basis of a possible dipolar model.     

Multi-Scale Computational Model of Fuel Homeostasis During Exercise: Effect of Hormonal Control

A mathematical model of the whole-body metabolism is developed to predict fuel homeostasis during exercise by using hormonal control over cellular metabolic processes. The whole body model is composed of seven tissue compartments: brain, heart, liver, GI (gastrointestinal) tract, skeletal muscle, adipose tissue, and “other tissues”. Each tissue compartment is described by dynamic mass balances and major cellular metabolic reactions. The glucagon–insulin controller is incorporated into the whole body model to predict hormonal changes during exercise. Moderate [150 W power output at 60% of peak oxygen consumption (VO_2max)] exercise for 60 min was implemented by increasing ATP utilization rates in heart and skeletal muscle. Arterial epinephrine level was given as an input function, which directly affects heart and skeletal muscle metabolism and indirectly other tissues via glucagon–insulin controller. Model simulations were validated with experimental data from human exercise studies. The exercise induced changes in hormonal signals modulated metabolic flux rates of different tissues in a coordinated way to achieve glucose homeostasis, demonstrating the efficacy of hormonal control over cellular metabolic processes. From experimental measurements of whole body glucose balance and arterial substrate concentrations, this model could predict the dynamic changes of hepatic glycogenolysis and gluconeogenesis, which are not easy to measure experimentally, suggesting the higher contribution of glycogenolysis (∼75%). In addition, it could provide dynamic information on the relative contribution of carbohydrates and lipids for fuel oxidation in skeletal muscle. Model simulations indicate that external fuel supplies from other tissue/organ systems to skeletal muscle become important for prolonged exercise emphasizing the significance of interaction among tissues. In conclusion, this model can be used as a valuable complement to experimental studies due to its ability to predict what is difficult to measure directly, and usefulness to provide information about dynamic behaviors.     

Simulation of intraluminal gas transport processes in the microcirculation

This study attempts to measure the hyperthermic response of individual microvessels in skeletal muscle tissue subject to local heating and then to predict the enhancement in thermal conductivity that results from the observed changes in vascular diameter and flow. In contrast to existing studies, which have tried to relate changes in tissue thermal conductivity to local blood perfusion using thermal clearance and self-heated thermistor techniques, we have developed a two-dimensional muscle tissue preparation in which the hyperemic response has been quantified by measuring thein vivo changes in diameter and blood flow of 1A to 4A generation vessels of rat cremaster muscle when the temperature was raised in 2° increments from 34 to 42°C. Only 3A and 4A vessels showed vasodilation when subject to hyperthermia, indicating that the measured increase in flow in the 1A and 2A vessels was the result of a decrease in downstream resistance. Our cremaster muscle preparations have also been used to obtain the first detailed anatomic measurements of the number density and length of countercurrent vessel pairs between 50–200 μm diameter. These combined measurements have been used to establish the limits of validity of the Weinbaum-Jiji theory. Our experimental data indicate that the Weinbaum-Jiji expression forK _eff is valid in cremaster muscle and cat mesentery tissue for both normal and hyperthermic conditions provided the largest vessels are <200 μm in diameter. The theory predicts that significant enhancements ink _eff start to occur for vessels that are 70 μm in diameter or larger, that a 2.5-fold increase ink _eff can be achieved for a maximally dilated 200 μm diameter 1A vessel pair in cremaster muscle of larger rats, and a 6-fold increase is predicted for maximally dilated 200 μm diameter vessels in the cat mesentery. The experiments also show that maximally dilated 1A to 4A vessels in the microcirculation closely satisfy the conditionQ(flow)/(2a)³=constant, which is consistent with the hypothesis that there is an adaptive regulation of vessel diameter which keeps the wall shear stress nearly constant during temporal changes in flow.     

Exhaustive exercise causes an anti-inflammatory effect in skeletal muscle and a pro-inflammatory effect in adipose tissue in rats

It is well known that exhaustive exercise increases serum and skeletal muscle IL-6 concentrations. However, the effect of exhaustive exercise on the concentrations of other cytokines in the muscle and in the adipose tissue is controversial. The purpose of this study was to evaluate the effect of exhaustive exercise on mRNA and protein expression of IL-10, TNF-α and IL-6 in different types of skeletal muscle (EDL, soleus) and in two different depots of white adipose tissue (mesenteric–MEAT and retroperitoneal–RPAT). Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6) and 6 (E6 group, n = 6) hours after the exhaustion protocol, which consisted of running on a treadmill (approximately 70% VO_2max for 50 min and then subsequently at an elevated rate that increased at 1 m/min every minute, until exhaustion). The control group (C group, n = 6) was not subjected to exercise. Cytokine protein expression increased in EDL, soleus, MEAT and RPAT from all exercised groups, as detected by ELISA. EDL IL-10 and TNF-α expression was higher than that of the soleus. The IL-10/TNF-α ratio was increased in the skeletal muscle, especially in EDL, but it was found to be decreased in the adipose tissue. These results show that exhaustive exercise presents a different effect depending on the tissue which is analysed: in the muscle, it induces an anti-inflammatory effect, especially in type 2 fibres, while the pro-inflammatory effect prevails in adipose tissue, possibly contributing to increased lipolysis to provide energy for the exercising muscle.     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.     

Functional roles of the gamma subunit of the skeletal muscle DHP-receptor

In excitation–contraction coupling (EC coupling) of skeletal muscle, large and rapid changes of the myoplasmic Ca²⁺ concentration mediate the activation and termination of force. The L-type Ca²⁺ channel (dihydropyridine receptor, DHP receptor) is a central component of the EC coupling process. Its predominant role is to provide the Ca²⁺ release channels of the sarcoplasmic reticulum (SR) with the sensitivity to cell membrane voltage. The DHP receptor consists of five different proteins (α_1S, β₁, γ₁, δ and α₂) whose tasks and functional characteristics are still incompletely understood. This short review summarizes progress made in studying the physiology of the γ₁ subunit, a membrane polypeptide that is highly specific for skeletal muscle. The focus is on recent results obtained from muscle of γ₁-deficient mice.     

Studies of the molecular mechanisms of action of relaxin on the adenylyl cyclase signaling system using synthetic peptides derived from the LGR7 relaxin receptor

The peptide hormone relaxin produces dose-dependent stimulation of adenylyl cyclase activity in rat tissues (striatum, cardiac and skeletal muscle) and the muscle tissues of invertebrates, i.e., the bivalve mollusk Anodonta cygnea and the earthworm Lumbricus terrestris, adenylyl cyclase stimulation being more marked in the rat striatum and cardiac muscle. Our studies of the type of relaxin receptor involved in mediating these actions of relaxin involved the first synthesis of peptides 619–629, 619–629-Lys(Palm), and 615–629, which are derivatives of the primary structure of the C-terminal part of the third cytoplasmic loop of the type 1 relaxin receptor (LGR7). Peptides 619–629-Lys(Palm) and 615–629 showed competitive inhibition of adenylyl cyclase stimulation by relaxin in rat striatum and cardiac muscle but had no effect on the action of relaxin in rat skeletal muscle or invertebrate muscle, which is evidence for the tissue and species specificity of their actions. On the one hand, this indicates involvement of the LGR7 receptor in mediating the adenylyl cyclase-stimulating action of relaxin in rat striatum and cardiac muscle and, on the other, demonstrates the existence of other adenylyl cyclase signal mechanisms for the actions of relaxin in rat skeletal muscle and invertebrate muscle, not involving LGR7 receptors. The adenylyl cyclase-stimulating effect of relaxin in the striatum and cardiac muscles was found to be decreased in the presence of C-terminal peptide 385–394 of the α_s subunit of the mammalian G protein and to be blocked by treatment of membranes with cholera toxin. These data provide evidence that in the striatum and cardiac muscle, relaxin stimulates adenylyl cyclase via the LGR7 receptor, this being functionally linked with G_s protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the G_s protein is mediated by interaction of the C-terminal half of the third cytoplasmic loop of the receptor with the C-terminal segment of the α_s subunit of the G protein. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 5, pp. 521–535, May, 2006.     

On the Mechanism of Antifibrillatory Effect of Afobazole

Effect of afobazole on the threshold of electrical fibrillation of the heart was studied on anesthetized rats with intact myocardium. It was shown that the drug considerably increased the threshold of electrical fi brillation of the heart, being not inferior to reference class I antiarrhythmic drugs (lidocaine and procainamide) according to V. Williamse classification. Against the background of preliminary injection of σ-receptor antagonist haloperidol, afobazole exhibited no antifibrillatory activity. These findings and analysis of published reports suggest that antifibrillatory activity of afobazole is determined by its antagonistic influence on σ₁-receptors localized in cardiomyocyte cytosol.     

Low frequency of the "plateau phenomenon" during maximal exercise in elite British athletes

A plateau in oxygen consumption (V̇O₂) has long been considered the criterion for maximal effort during an incremental exercise test. But, surprisingly, the termination of a maximum exercise test often occurs in the absence of a V̇O₂ plateau. To explain this inconsistency, some have proposed that an oxygen limitation in skeletal muscle occurs only in elite athletes. To evaluate this hypothesis, we determined the frequency with which the "plateau phenomenon" developed in a group of elite male and female athletes. Fifty subjects performed a continuous incremental treadmill test to measure maximal oxygen consumption (V̇O_2max). Treadmill velocity increased by 0.31 m s⁻¹ until the respiratory exchange ratio (R) reached 1.00. Thereafter the treadmill gradient increased by 1% each minute until exhaustion. The V̇O_2max was the highest V̇O₂ sustained for 60 s. Three criteria were used to determine maximal efforts: (1) a plateau in the V̇O₂, defined as an increase of less than 1.5 ml kg⁻¹ min⁻¹; (2) a final R of 1.1 or above; (3) a final heart rate (HR) above 95% of the age-related maximum. Mean V̇O_2max exceeded 65 ml kg⁻¹ min⁻¹ in both groups. The criteria for R and HR were satisfied by 72% of males and 56% females, and 55% of males and 69% of females, respectively. In contrast a V̇O₂ plateau was identified in only 39% of males and 25% of females. These findings refute the twin arguments: (1) that the absence of a "plateau phenomenon" results from an inadequate motivational effort in poorly trained athletes and (2) that the "plateau phenomenon" and a consequent skeletal muscle anaerobiosis occur only in athletes with the highest V̇O_2max values.     

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells

 The properties of the calcium stores coupled to a depletion-operated cation current (I _DOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 μM) activated an initial, large (≈ 200 pA), transient, calcium-dependent chloride current (I _ClCa) followed by a smaller (≈ 10 pA) sustained, non-selective cation current (I _DOC). Intracellular application of heparin (5 mg ml^–1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 μM) activated I _DOC but not I _ClCa; ryanodine (30 μM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of I _ClCa by carbachol. In the presence of 30 μM, but not 3 μM, ryanodine, carbachol was able to activate I _DOC. Cyclopiazonic acid (CPA; 10 μM) abolished the response to carbachol; however, caffeine was still able to activate I _ClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 μM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4,5-trisphosphate-(IP₃-) sensitive or ryanodine-sensitive calcium stores is able to activate I _DOC, and, by extension, capacitative calcium entry in this tissue. Received: 21 July 1997 / Received after revision: 29 August 1997 / Accepted: 1 September 1997     

Prediction of Extravascular Burden of Carbon Monoxide (CO) in the Human Heart

Clinically significant myocardial abnormalities (e.g., arrhythmias, S-T elevation) occur in patients with mild-to-severe carbon monoxide (CO) poisoning. We enhanced our previous whole body model [Bruce, E. N., M. C. Bruce, and K. Erupaka. Prediction of the rate of uptake of carbon monoxide from blood by extravascular tissues. Respir. Physiol. Neurobiol. 161(2):142–159, 2008] by adding a cardiac compartment (containing three vascular and two tissue subcompartments differing in capillary density) to predict myocardial carboxymyoglobin (MbCO) and oxygen tensions (P_cO₂) for several CO exposure regimens at rest and during exercise. Model predictions were validated with experimental data in normoxia, hypoxia, and hyperoxia. We simulated exposure at rest to 6462 ppm CO (10 min) and to 265 ppm CO (480 min), and during three levels of exercise at 20% HbCO. We compared responses of carboxyhemoglobin (HbCO), MbCO and P_cO₂ to estimate the potential for myocardial injury due to CO hypoxia. Simulation results predict that during CO exposures and subsequent therapies, cardiac tissue has higher MbCO levels and lower P_cO₂’s than skeletal muscle. CO exposure during exercise further decreases P_cO₂ from resting levels. We conclude that in rest and moderate exercise, the myocardium is at greater risk for hypoxic injury than skeletal muscle during the course of CO exposure and washout. Because the model can predict CO uptake and distribution in human myocardium, it could be a tool to estimate the potential for hypoxic myocardial injury and facilitate therapeutic intervention.     

Insertion of α1S II–III loop and C terminal sequences into α1H fails to restore excitation–contraction coupling in dysgenic myotubes

The L-type Ca²⁺ channel in skeletal muscle (α_1S) is essential for excitation–contraction (EC) coupling. Previous studies using chimeras composed of α_1S together with α_1C or α_1M demonstrated the importance of the α_1S II–III loop and of a smaller subdomain (residues 720–764; ‘ECC’) in skeletal EC coupling. However, these chimeras failed to test the significance of regions outside the II–III loop, which are highly conserved between α_1S and α_1C. Therefore, we have injected dysgenic (α_1S-lacking) myotubes with cDNAs encoding chimeras between α_1S and the highly divergent T-type Ca²⁺ channel, α_1H. The chimeras consisted of GFP-tagged α_1H with one or more of the following substitutions: α_1S II–III loop residues 720–764 (‘ECC’), a putative targeting domain of the α_1S C terminus (‘target’; residues 1543–1662) or the entire α_1S C terminus (‘Cterm’; residues 1382–1873). The presence of either target or Cterm affected the expression and/or kinetics of whole-cell currents recorded from both dysgenic muscle cells and tsa-201 cells. Importantly, substitution of ECC alone into GFP-α_1H (GFP-α_1H + ECC), or together with either target (GFP-α_1H + ECC + target) or Cterm (GFP-α_1H + ECC + Cterm ), was insufficient to restore electrically evoked contractions. Depolarization-induced fluorescence transients for GFP-α_1H + ECC, GFP-α_1H + ECC + target or GFP-α_1H + ECC + Cterm had a bell shaped dependence upon membrane voltage (inconsistent with skeletal EC coupling) and were also exceedingly small (unlike cardiac EC coupling). The absence of EC coupling for these chimeras raises the possibility that regions of α_1S outside of ECC and target are necessary for providing the context that allows these two domains to function in EC coupling and targeting, respectively. Additionally, an inadequate membrane density of the chimeras may have contributed to the lack of coupling. This revised version was published online in July 2006 with corrections to the Cover Date.