by Suzanna Engman

IN THEIR EXPERIMENTS on a variety of organisms, from tadpoles to electric sting rays and cultured chick muscle cells to transgenic mice, biophysicist José A. Lasalde-Dominicci, Ph.D., University of Puerto Rico, Río Piedras and his teams of researchers study just about every aspect of nicotinic acetylcholine receptors. They’re focusing on the interrelationship of the receptors’ proteins with the lipid membrane, how interactions are regulated, and how mutations in these receptors lead to disease. Currently, Lasalde is working on four NIH grant-funded projects investigating the various roles of these receptors. The projects involve both laboratory and clinical research in collaboration with the University of Minnesota and Scripps Institute.

“It is our hope that these studies will identify potential therapeutic targets or positive allosteric agents [agents that alter the protein’s activity by combining with another substance at a point other than the chemically active site] that might benefit a broader range of neurodegenerative disorders,” says Lasalde. His ultimate goal is to crystallize the nicotinic receptor, which would reveal its structure in high resolution and facilitate acetylcholine receptor drug research and development.

Studying the nicotinic acetylcholine receptor (nAChR) function using biochemical, biophysical, and molecular approaches will help Lasalde and other researchers to understand the pathophysiology of nicotinic receptor–related diseases such as Alzheimer’s, schizophrenia, congenital myasthenic syndrome, epilepsy, Parkinson’s, and nicotine addiction. While some nicotinic drugs have been developed to treat these diseases, for example Alzheimer’s drugs deliver a series of ACh inhibitors, their therapeutic effects are limited. Alzheimer’s drugs lose their effectiveness after two or three years; the receptors become desensitized, and neurons start to die.

Recent findings suggest that schizophrenic patients have a defect in the nAChRs of the central nervous system that causes abnormal sensory gating. Nicotine use improves sensory gating temporarily among these patients. “There is a paradox of chronic smoking and schizophrenia. Ninety percent of schizophrenics are chronic smokers because nicotine for them is a form of automedication—it provides relief from the symptoms. Chronic smoking elevates the number of nicotinic receptors, up to three to five times that of the nonsmoking population and this is directly related to nicotine addiction. However, chronic schizophrenic smokers don’t have the elevated numbers of nicotinic receptors. These findings suggest that there’s a nicotinic deficit in schizophrenia.”

NICOTINIC ACETYLCHOLINE RECEPTORS Nicotinic acetylcholine receptors are located throughout the body as neuronal receptors in the central and peripheral nervous system, and neuromuscular receptors in the neuromuscular junctions of somatic muscles. They affect almost every human function, including breathing, movement, cognition, memory, and mood. The receptors form ion channels, protein-lined pores that control the flow of ions across the membranes of all living cells. Once the extracellular domain has been activated by the binding of the appropriate ligand, the pore becomes accessible to ions, which then pass through. The opening of the receptor is gated by the neurotransmitter acetylcholine (ACh) and by nicotine. In other words, both ACh and nicotine are able to open the channel, allowing positively charged ions—in particular sodium and calcium—to enter the cell membrane. Nicotinic receptors are made up of five subunits, arranged symmetrically around the ion channel.

WHAT IS CRYSTALLIZATION? A crystal is a solid in which the constituent atoms, molecules, or ions are packed in a regularly ordered, repeating pattern. Some materials, for example, proteins, do not occur naturally as crystals, but scientists can create a protein crystal by placing the protein in a solution and allowing it to crystallize over a period of time via vapor diffusion. A drop of solution containing the molecule, buffer, and precipitants is sealed in a container with a reservoir containing a hygroscopic solution. Water in the drop diffuses to the reservoir, slowly increasing the protein concentration and allowing a crystal to form. Once a crystal is formed, scientists can indirectly, through mathematical modeling, study its molecular structure. The double–helical structure of DNA was deduced from crystallographic data. Crystallization of a protein is very difficult because proteins are fragile, sensitive to heat and light. In addition, the molecules need time to align themselves properly at the surface of a growing crystal in order for the crystal to continue to grow regularly. If the solution becomes over–saturated so that the solid forms too quickly, the molecules do not have time to align properly, and the crystal is irregularly formed.

One of the projects in Lasalde’s laboratory relates to the nicotine-induced upregulation of neuronal nicotinic receptors. “This research will uncover mechanisms involved in responses to chronic nicotinic ligand exposure that relate to nicotine tolerance, dependence, and withdrawal. This knowledge could impact treatment of tobacco use and help eliminate or control a major cause of death and disability.”

Another of Lasalde’s projects focuses on the nAChR’s role in slow channel congenital myasthenic syndrome (SCCMS). Congenital myasthenic syndrome is an inherited disorder that causes muscle weakness (myasthenia) by affecting the connection between nerve cells and muscle cells. SCCMS is caused by extended opening of the AChR, which overstimulates the muscles.

The inheritance pattern for most types of CMS is autosomal recessive, meaning that it takes two copies of the defective gene (one from each parent) to cause the disease. However, SCCMS is inherited in an autosomal dominant manner; in other words, one copy of a defective AChR gene is enough to cause the disease, so an affected parent has a 50 percent chance of passing the disease on to a child.

Lasalde’s group, in collaboration with Dr. Christopher Gómez at the University of Minnesota and Dr. Legier Rojas at UCC Bayamón, investigates nicotinic neuromuscular receptor genes of patients with SCCMS. The researchers have developed transgenic mice models that contain the same genetic mutation as humans with SCCMS to identify the mechanism that causes muscle fiber degeneration. Most of the students in Lasalde’s laboratory use state-of-the-art electrophysiological techniques to record the electric activity of the ion channel receptors in the recombinant expression system or in muscle fibers. They use confocal and two photon microscopy (www.cifupr.org) to observe whether calcium is moving correctly in the muscle neuromuscular junction.

Torpedo californica Electric Ray

The researchers also observe the phenotypical expression of the disease in the transgenic mice by testing the strength of their muscles using a mouse-sized laboratory tightrope. A healthy mouse or a mouse that carries an unexpressed SCCMS genetic defect can balance on the tightrope, but a mouse expressing SCCMS will fall.

“In seven or eight family members with the same SCCMS mutation, some may show no signs of the disease, others will develop it at an early age, and others will develop it at a later age. We’re studying what triggers the phenotype. The use of transgenic mice models for SCCMS mutations will help clarify the pathogenesis of this disease and identify mechanisms relevant to other central nervous system diseases.”

Lasalde’s ultimate goal is to accomplish one of the most difficult tasks in biochemistry today—crystallizing a membrane protein, the nicotinic receptor. For the last fifteen years, he has been developing the methodology for growing a membrane protein crystal. “We work from natural sources, for example the Torpedo californica Electric Ray, commonly known as the sting ray, to find ways to make the protein crystallize.

“Since 1997, our laboratory has screened approximately 300,000 crystallization conditions using AChR purified from the Torpedo electric organ from T. californica and T. nobiliana. We established a collaborative research agreement with Dr. Raymond Stevens at the Scripps Research Institute in San Diego, CA. The work is done in close collaboration with the Stevens laboratory at TSRI where Guillermo Asmar (M.S. from UPR) is working. This collaboration enables critical access to the Joint Center for Innovative Membrane Protein Technologies, as well as other high throughput crystallization, imaging, and synchrotron beam time. During this period, we have gathered an enormous amount of data and very encouraging results.

“The biggest problem in biochemistry now is membrane proteins. No remarkable progress has been made in the last twenty years. It is difficult to purify—almost impossible to crystallize. For fifteen years we’ve been working to crystallize the nicotinic receptor. If you crystallize it, you can see its structure at high resolution. We have made progress, but it is very difficult. This is a large protein with 2,333 amino acids.”

Designing the methodology for something that has never been done before, researching four grant-funded research projects, overseeing 38 researchers, teaching, and attending study sections at the NIH doesn’t leave much time for recreation, but Lasalde says that his work offers compensatory rewards. “In the last eight years my laboratory has contributed to the formation of about 30 Ph.Ds. Some of them have graduated from UPR and others have trained for two or three years in my laboratory and then joined top graduate schools on the mainland, for example Harvard, Berkeley, UCLA, Cornell, and Baylor.

“At about 14 or 15 I decided to become a scientist. I liked chemistry and math. I knew that math was essential to becoming a good chemist. I like chemistry because it’s fun—it’s fun to solve a biophysical problem, but the best part of my job is that it provides the space and time to be creative.”

jseal@coqui.net