The Na-Ca exchanger is the dominant sarcolemmal calcium extrusion mechanism in heart muscle. Despite its importance, only recently have quantitative investigations into its function been carried out. In the proposed experiments, biophysical, biochemical and pharmacologic properties of the Na-Ca exchanger will be examined. The goal of this work is to determine how the Na-Ca exchanger works in heart muscle, how it is modulated by important cations (e.g. Ca2+, Na+, H+) and metabolic factors (e.g. ATP) and how it may be altered by drugs and inorganic inhibitors. Additionally, the planned work will examine specific features of the Na-Ca exchanger to determine how it influences contraction in heart. The proposed experiments will make use of two important new approaches for investigations of the Na-Ca exchanger and will use isolated guinea pig and rat heart cells. (1) The first method has been developed in the PI's laboratory. By integrating a flashlamp into the voltage-clamp fluorescence microscopy system, caged calcium (DM nitrophen and Nitr-5) can be used to produce [Ca2+]i jumps to activate the Na-Ca exchanger. Two components of the Na-Ca exchanger current have been identified. One component (never before seen) arises from a molecular conformational change of the transport protein (Iconf) while the other reflects net transport by the exchanger (INa-Ca). Quantitative data generated by this method is quite novel, having been unobtainable until now and represents an important new source of information about the exchanger in functioning cells and will be used as a new tool in the proposed experiments. Preliminary experiments have been carried out using this method to provide estimates of the density of exchangers in the sarcolemma, the turnover rate of the exchanger under various conditions and the charge on the """"""""naked"""""""" exchanger protein (i.e. without sodium or calcium bound). The novelty and power of this method is further enhanced because cell length and [Ca2+}i, or [Na+]i or pHi (using fluorescent indicators) can be measured simultaneously. This method will therefore be used to investigate the properties of the Na-Ca exchanger and to examine the effects of exchanger activity on contraction in heart cells. (2) The second approach uses """"""""giant"""""""" sarcolemmal membrane patches to measure Na-Ca exchanger current (Hilgemann, 1989, 1990) providing access to both intracellular and extracellular surfaces of the sarcolemmal membrane while measuring INa-Ca. Specific experiments using the above methods are designed to address two broad questions. (1) How is the Na-Ca exchanger controlled within the heart cell? (2) How does the Na-Ca exchanger participate in excitation- contraction coupling? The proposed work should provide important primary information that will broaden our understanding of how the Na-Ca exchanger functions and how it influences [Ca2+]i SR calcium loading and EC coupling in heart muscle. Accordingly, the planned investigation should provide us with new understanding on the normal and pathologic functioning of the heart.
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