The rod outer segment sodium-calcium exchanger

Debora A Nicoll, Purdue University

Abstract

To be able to further our understanding of the role of Ca$\sp{2+}$ in the rod outer segment (ROS) during signal transduction, some of the properties of the Na$\sp+$/Ca$\sp{2+}$ exchanger, one of the major Ca$\sp{2+}$-transporting proteins of the ROS, in a crude, reconstituted preparation have been examined, the conditions needed to maintain exchanger activity during purification have been determined and the exchanger purified. The characteristics of Ca$\sp{2+}$-uptake by the ROS exchanger have been studied by solubilizing and reconstituting the ROS proteins into liposomes and measuring Ca$\sp{2+}$-fluxes into the liposomes. Additions of Ca$\sp{2+}$- or Na$\sp+$ ionophores indicate that Ca$\sp{2+}$ is taken up into the liposomes rather than bound to sites on the membrane, and that a Na$\sp+$-gradient seems to be required to drive the exchanger reaction. The ion requirements of the exchanger have also been determined: K$\sp+\sb{\rm out}$ (K$\sb{\rm m}$ = 2mM), Ca$\sp{2+}\sb{\rm out}$ (K$\sb{\rm m}$ = 1.3$\mu$M) and Na$\sp+\sb{\rm in}$ (K$\sb{\rm m}$ = 71mM) are required to drive the exchanger while Na$\sp+\sb{\rm out}$ (K$\sb{\rm i}$ = 16mM) inhibits. The exchanger appears to act in an electrogenic manner. The conditions needed to maintain exchanger activity during purification were determined to be: 10 mM Cholate or 7.5 mM CHAPS, pH $\geq$ 7, NaCl $\geq$ 50 mM, and 0.1% soybean asolectin. It was also found that addition of the sulfhydryl modifying reagent, dithiothreitol, to the solubilization solution caused a time-dependent loss of exchanger activity. Using the above conditions, the exchanger was purified 336-fold by a combination of solubilization, and ion-exchange and wheat germ agglutinin chromatography. Activity from these columns most closely correlates with the presence of a polypeptide of MW 215 kDa. As observed for the ROS exchanger in crude preparations, the purified exchanger also requires K$\sp+$ for activity.

Degree

Ph.D.

Advisors

Applebury, Purdue University.

Subject Area

Biochemistry

COinS