I worked with several mentors while I was a graduate student at the University of Virginia. During a rotation in the laboratory of Howard Kutchai, I pursued the isolation of the 53kDa glycoprotein (GP-53) of the sarcoplasmic reticulum (SR) thought to regulate the SR Ca-ATPase. The 53kDa glycoprotein is a major protein in the SR membrane and that had been co-localized with the Ca-ATPase in at a constant molar ratio of 2GP-53 : 3Ca-ATPase and to be expressed concurrently with the Ca-ATPase during myogenesis. Using biochemical approaches we strived to isolate it and learn more about its function. During my rotation, I used a concavalin-A column to isolate glycoproteins from a rabbit S.R. preparation. The Con-A column worked reasonably well in the isolation of a crude fraction composed primarily of GP-53. Dr. Kutchai taught me biochemistry techniques, how to think like a researcher, and how to juggle multiple projects. He continued to mentor me throughout my graduate years.

I did my thesis project under the primary mentorship of Kunio Takeyasu. Dr. Takeyasu had just completed a postdoc in Doug Fambrough's laboratory at Johns Hopkins University and he brought with him stellar skills in molecular biology and an interest in the Na/K-ATPase (a relative of the Ca-ATPase I had been studying). I knew I had to learn the powerful techniques of molecular biology and joined his lab. I had an excellent experience as a student with Kunio as my primary professor and the added mentorship of Howard Kutchai and later Guiseppe Inesi (University of Maryland at Baltimore). Kunio was my source for molecular biology methodology and Na/K-ATPase information while Howard and Guiseppe were my sources for biochemical methodology and S.R. Ca-ATPase information. My dissertation was entitled: "The molecular dissection of the ligand binding domains of the Na/K-ATPase and the S.R. Ca-ATPase." Molecular biology allowed me to perform powerful manipulations of these pumps and biochemistry provided the tools to study the result. Over the five years I worked on my thesis I was successful in publishing three papers about our findings (Luckie et al. 1991, 1991, 1992).
 

DISSERTATION RESEARCH SUMMARY:

The Na⁺/K⁺-ATPase (sodium pump) is a multi-subunit integral membrane protein thought to be comprised of one alpha and one beta subunit. This ouabain (cardiac glycoside)-inhibitable enzyme couples the free energy of one ATP molecule to translocate three sodium ions out of the cell and two potassium ions in. The alpha subunit (approximately 100kD) is the catalytic subunit (splits ATP) and it appears to contain the ouabain-binding site(s) as well as the binding sites of the sodium and potassium ions. Our laboratory suspected beta played a role in transporting or stabilizing the alpha subunit, and that it might also have been involved in ouabain binding. The Ca2+-ATPase of the sarcoplasmic reticulum is quite homologous to the Na/K-ATPase (both members of the P-type ATPases). The calcium pump is a ~100kD protein with primary and secondary structure like the sodium pump's alpha-subunit. Since the amino acid sequences between the sodium-pump alpha-subunit and the calcium-pump were well conserved, and the hydrophobicity plots were very similar, we hypothesized that the general structures of both ion-pumps were the same. My goal was to identify the functional domains that give the Na/K-ATPase and Ca-ATPase their specificity for particular ions, inhibitors, and cofactors (beta). My approach was to construct chimeric (hydrid) molecules between these functionally different but structurally similar cation ATPases. In order to achieve this, I took advantage of an identical region between the two pumps (in exon 16) and as the exchange point and used a unique restriction enzyme site (Eco N1 site) to cut and recombine the genes. An chicken sodium pump alpha subunit cDNA and calcium-pump cDNA were cross-ligated at the EcoNI site and the resulting chimeric cDNAs were transfected (with a neomycin resistance plasmid) into mouse L cells. The L cells had also been previously been transfected with a chicken sodium pump beta-subunit cDNA.

Here's an example of the "power" of the molecular biology we used. By placing both 'neomycin-resistance' and 'chicken' genes in our mouse L-cells, we could first screen the cells with G418 (neomycin) and isolate the resistant cells (the ones that took up our cDNAs). Then we could search with chicken specific monoclonal antibodies to find which mouse cells expressed chicken proteins (mouse cells usually only express mouse proteins). As a result we successfully cloned several stable cell lines that expressed both chicken beta-subunit and our chimeric molecules. Once we had the cells lines, the expression, subcellular distribution and function of the chimeric proteins were studied with various biochemical approaches in the Takeyasu, Kutchai and Inesi laboratories.

Chimeric molecule I (acronym NNC), consisting of the N-terminal 2/3 of the plasma membrane Na-pump and the C-terminal 1/3 of the SR Ca-pump, was found to be expressed exclusively inside the cells, did not assemble with a beta subunit, but exhibited ouabain-sensitive ATPase activity which could be stimulated by Ca ions. It could bind ouabain and Ca ions with high affinity equal to the wild-type pumps. Chimeric molecule II (CCN), containing the N-terminal 2/3 of Ca-pump and the C-terminal 1/3 of the Na-pump, was found to reside on the plasma membrane as well as inside the cells, assemble with the Na-pump beta subunit, and showed no Ca stimulated or ouabain-sensitive ATPase activity.

As a result of our research some of the critical domains for P-type ATPase function identified in these chimeric studies were: (i) The targeting and assembly domains on the chicken Na/K-ATPase alpha subunit were localized to residues 713-1014, (ii) The ouabain binding domain was localized in the alpha subunit among residues 93-147 and 290-346, (iii) The high affinity Ca binding sites of the sarcoplasmic reticulum Ca-ATPase were localized to residues 723-994. My research also showed the 'chimeric recombinant technique' was a valid approach in the delineation of functional domains when applied between proteins of conserved structure but divergent function.

After working with membrane transporters at UVA I wanted to apply my skills to proteins associated with clinical maladies. I also wanted to learn patch clamp and powerful electrophysiology approaches used to study ion channels. I found a perfect match in the Cystic Fibrosis Research Laboratory run by Jeffrey Wine at Stanford University.
 
 

References:

D.B. Luckie, K.L. Boyd, A. Mizushima, Z. Shao, A. Somlyo and K. Takeyasu (1991) Identification of ouabain-binding and Ca-stimulation domains in Na- and Ca-pump chimeric molecules. in The Sodium Pump: Recent Developments, Rockfeller Univ. Press NY, p.237-242.

D.B. Luckie, K.L. Boyd, and K. Takeyasu (1991) Ouabain and Ca2+-sensitive ATPase activity of chimeric Na- and Ca-pump molecules. FEBS letters, 281:231-234.

D.B. Luckie, V. Lemas, K.L. Boyd, D.M. Fambrough, and K. Takeyasu (1992) Molecular dissection of functional domains of the E1E2ATPases using sodium and calcium pump chimeric molecules. Biophys. J., 62:227-234.