Work in the lab focuses on Ca2+ signaling in living cells. By combining confocal, two photon or wide-field microscopy imaging with whole cell patch clamp techniques, we have been able to investigate the effects of local and global [Ca2+]I on cellular function. Diverse additional tools are used as needed including flash photolysis of caged chemicals, multi-photon uncaging, single channel examination in planar lipid bilayer and by patch clamp, immunofluorescence imaging, use of cells from transgeinc and gene knockout animals, use of primary cultures and co-cultures. There are five areas of active work.
1. Cellular and Molecular Ca2+ signaling.
Local Ca2+ signals depends on the subcellualr organization of the affected cells. Ryanodine receptors (RyRs) and IP3 receptors (IP3Rs) are opened by an increase in [Ca2+]I. This local increase in [Ca2+]I may come from Ca2+ channels on the plasmalemmal membrane, that Na+/Ca2+ exchanger bringing Ca2+ into the cell, non-selective or poorly selective cation channels, or other intracellular Ca2+ release channels. Recently we have identified a new source of such activator Ca2+:Na+ channels whose selectivity is transiently modified. Specific activators and kinases modulate both the sources of Ca2+ influx and the RyRs and IP3Rs. In many cells, there are subcellular regions in which Ca2+ - conducting channels to be amplified. The RyRs and IP3Rs are intracellylar Ca2+ release channels with large conductances that are found in the sarcoplasmic reticulum and in the endoplasmic reticulum of many cells.
2. Heart Cells.
In heart cells, Ca2+ sparks reflect the Ca2+ -dependent activation of RyRs. Ca2+ sparks are the elementary events in exitation-contraction coupling. The activation of Ca2+ sparks by the L-type Ca2+ channel current (and more recently Ca2+ flux through Na+ channels) underlies normal contraction. Under normal pathalogic conditions, arrhythmais can occur due to the untowards activiation of Ca2+ sparks. In heart failure, the inadequate activation of Ca2+ sparks can explain, in part, the pathology. Current work is examining how SR Ca2+ release is activated and how it is terminated.
3. Smooth Muscle Cells.
Arterial smooth muscle contains Ca2+ -activated potassium channels and both RyRs and IP3Rs. The activation of CA2+ sparks in such smooth muscle (due to the opening of RyRs) activates the Kca channels and leads to hyperpolarization of the smooth muscle. By this means, an increase n local [Ca2+]I activates cell-wide relaxation. Much of this work was carried out using cascular smooth muscle from brain arteries. Similar results, however, occur in the coronary arteries. Current work examines how Ca2+ sparks are modulated in smooth muscle and how Ip3Rs interact with RyRs.
4. Skeletal muscle cells.
Since voltage-gated SR Ca2+ release occurs in mammalian skeletal muscle, it was surprising that we could observe Ca2+ sparks in these cells due to two processes. We observed CA2+ sparks activated directly by depolarization. In addition, we observed CA2+ sparks activated by a local increase in [Ca2+]I. Current work examines how the local [Ca2+]I signals change with development and how the developmental regulation of the transverse tubules affects Ca2+ signaling.
[Ca2+]I signals in neurons appears to be more restricted in size than in muscle. Furthermore, there appears to be a larger role played by the IP3Rs. The more diverse kinds of Ca2+ channels available, the additional sources of CA2+ influx and efflux contribute to the complex regulation of local [Ca2+]I signals in neurons. Additionally, the role of local [Ca2+]I signals in influencing both pre-synaptic and post-synaptic signaling are being studied. Work has centered on an examination of DRG neurons, on cereballar Purkinje cells and on other CNS neurons that display Ca2+ -induced CA2+ -release.
Santana, L.F., Kranias, E.G., and Lederer, W.J. (1997) Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocytes. J. of Physiol., 503: 21-29.
Santana, L.F., Gomez, A.M. and Lederer, W.J. (1998) Ca2+ flux through promiscuous cardiac Na+ channels: Slip-mode conductance. Science, 279: 1027-1033.