The interaction of an impermeant cation with the sheep cardiac RyR channel alters ryanoid association.

In previous studies, we have demonstrated that the interaction of ryanoids with the sarcoplasmic reticulum Ca(2+)-release channel [ryanodine receptor (RyR)] incorporated into planar lipid bilayers reduced the effectiveness of tetraethylammonium (TEA(+)) as a blocker of K(+) translocation (J Gen Physiol 117: 385-393, 2001). In the current study, we investigated both the effect of TEA(+) on [(3)H]ryanodine binding and the actions of this impermeant cation on the interaction of the reversible ryanoid 21-amino-9alpha-hydroxyryanodine with individual, voltage-clamped RyR channels. A dose-dependent inhibition of [(3)H]ryanodine binding was observed in the presence of TEA(+), suggesting that the cation and alkaloid compete for access to a common site of interaction. Single channel studies gave further insights into the mechanism of the competition between the two classes of ligands. TEA(+) decreases the association rate of 21-amino-9alpha-hydroxyryanodine with its receptor, whereas the dissociation rate of the ryanoid from the channel was unaffected. Our results demonstrate that TEA(+) inhibits both K(+) translocation through RyR, and ryanoid interaction at the high affinity ryanodine site on the channel. These actions involve binding of TEA(+) to different, but weakly interacting, sites in the RyR channel.

Voltage-sensitive equilibrium between two states within a ryanoid-modified conductance state of the ryanodine receptor channel.

We have investigated the influence of transmembrane holding potential on the kinetics of interaction of a cationic ryanoid, 8beta-amino-9alpha-hydroxyryanodine, with individual ryanodine receptor (RyR) channels and on the functional consequences of this interaction. In agreement with previous studies involving cationic, neutral, and anionic ryanoids, both rates of association and dissociation of the ligand are sensitive to transmembrane potential. A voltage-sensitive equilibrium between high- and low-affinity forms of the receptor underlies alterations in rates of association and dissociation of the ryanoid. The interaction of 8beta-amino-9alpha-hydroxyryanodine with RyR influences the rate of cation translocation through the channel. With this ryanoid bound, the channel fluctuates between two clearly resolved subconductance states (alpha and beta). We interpret this observation as indicating that with 8beta-amino-9alpha-hydroxyryanodine bound, the pore of the RyR channel exists in two essentially isoenergetic conformations with differing ion-handling properties. The equilibrium between the alpha- and beta-states of the RyR-8beta-amino-9alpha-hydroxyryanodine complex is sensitive to transmembrane potential. However, the mechanisms determining this equilibrium differ from those responsible for the voltage-sensitive equilibrium between high- and low-affinity forms of the receptor.

Human mesenchymal stem cells form Purkinje fibers in fetal sheep heart.

BACKGROUND: We have investigated the usefulness of a model of cardiac development in a large mammal, sheep, for studies of engraftment of human stem cells in the heart. METHODS AND RESULTS: Adult and fetal human mesenchymal stem cells were injected intraperitoneally into sheep fetuses in utero. Hearts at late fetal development were analyzed for engraftment of human cells. The majority of the engrafted cells of human origin formed segments of Purkinje fibers containing exclusively human cells. There were no differences in engraftment of human mesenchymal stem cells from adult bone marrow, fetal brain, and fetal liver. On average, 43.2% of the total Purkinje fibers in random areas (n=11) of both ventricles were of human origin. In contrast, approximately 0.01% of cardiomyocytes were of human origin. CONCLUSIONS: Human mesenchymal stem cells preferentially engraft at high levels in the ventricular conduction system during fetal development in sheep. These findings raise the possibility that stem cells contribute to normal development of the fetal heart.

Imaging single cardiac ryanodine receptor Ca2+ fluxes in lipid bilayers.

In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca2+ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca2+ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca2+ (25-50 mM) that result in saturating RyR2 Ca2+ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca2+ currents having amplitudes of 0.25-14 pA. Ca2+ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca2+ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca2+ currents, and for addressing other current research questions is discussed.

An anionic ryanoid, 10-O-succinoylryanodol, provides insights into the mechanisms governing the interaction of ryanoids and the subsequent altered function of ryanodine-receptor channels.

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.

Excess noise in modified conductance states following the interaction of ryanoids with cardiac ryanodine receptor channels.

The interaction of ryanodine with the ryanodine receptor (RyR) produces profound changes in channel function. Open probability increases dramatically and conductance is reduced. In this report we describe differences in the properties of reduced conductance states produced by the interaction of ryanodine derivatives with RyR channels. Some reduced conductance states are considerably noisier than the normal open state of the RyR channel. Inspection and analysis of these events reveals that the excess noise arises from transitions between two conductance states. Following the interaction of certain ryanodine derivatives, RyR channels undergo transitions between two conformations with slightly different ion-handling properties.