May 15, 2000 EVANSTON, Ill. -- A new Northwestern University study that advances the understanding of the genetics of hearing disorders is reported in the May 11 issue of the journal Nature. In an unusual collaboration of hearing science and molecular biology experts, researchers have cloned a gene, named after the musical notation presto, that is critical to the functioning of the outer hair cell, a sensory receptor cell unique to the inner ear of mammals.
Research teams under the direction of Peter Dallos, John Evans Professor of Neuroscience, and Laird D. Madison, M.D., assistant professor at the Center for Endocrinology, Metabolism and Medicine at the University's Medical School, have successfully cloned the gene Prestin which codes for a protein (prestin) that is an important molecular motor in the outer hair cell. Prestin is a new biological motor in mammals, joining the classical molecular motors kinesin, dynein and myosin, the latter being the protein that causes muscles to contract.
Roughly 25 million people in the United States are severely hard of hearing, and many of these deficits are related to the loss of outer hair cell function. In addition to the health implications of the prestin discovery, these tiny biocompatible motors could be a boon to the developing nanotechnology field.
"This is a landmark study," said Mario Ruggero, Hugh Knowles Professor of Hearing Sciences and head of the audiology and hearing sciences program, department of communication sciences and disorders, at Northwestern. "It identifies the molecular basis for the motile process that makes the outer hair cells of the mammalian cochlea unique and which may be responsible for the exquisite sensitivity of sound perception in humans."
It is widely believed that outer hair cells act as local mechanical amplifiers of the incoming sound vibrations, giving the mammalian ear its extraordinary sensitivity and frequency-resolving capacity. During amplification, the cylinder-shaped outer hair cells elongate and contract at the same very rapid rate as the frequency of the incoming sound. They boost the signals received by the inner hair cells, the sensory cells located in the cochlea that are responsible for communicating with the central nervous system. The inner hair cells send the auditory information to the brain, which interprets it as a teakettle's whistle or a Puccini aria.
"We strongly believed that a molecular motor was responsible for the changes in the cell's length, so we set out to find the motor molecule that is so critical to hearing in mammals," said Dallos, also Hugh Knowles Professor of Audiology and Hearing Sciences. "What is most amazing about this motor is its speed. It moves faster than anything else we know in the human body."
Human beings can hear sounds whose frequencies range from a rumble to a squeak, from approximately 20 to 20,000 Hertz (cycles per second). Small mammals, mice for example, can hear up to 60,000 Hz. That means that a high squeak might cause the outer hair cells to alter their length as often as 60,000 times a second. Noise trauma, due to loud music or a nearby explosion, for example, can damage or destroy outer hair cells, of which mammals have a fixed number established near birth. Without outer hair cells, amplification cannot occur and a mammal's auditory abilities are greatly diminished, leaving it with a primitive form of hearing.
In addition to its speed, the other unique feature of the prestin motor is that it does not require extra biological energy, such as adenosine triphosphate (ATP), to function. Instead, the incoming sound stimulus produces electrical voltage changes in the outer hair cells that drive the prestin motor. Unlike prestin, the functioning of the other biological motors, kinesin, dynein and myosin, do require ATP, the major energy molecule found in cells.
In the hunt for the gene responsible for the outer hair cell's motor protein, the Dallos-Madison teams analyzed the genetic makeup of the outer and inner hair cells of gerbils. These two types of cells are very similar to each other, except for certain proteins unique to outer hair cells. Inner hair cells are not motile.
"The real difficulty during this process was that we had a limited amount of material with which to work," said Madison. "We needed to isolate approximately a thousand each of the inner and outer hair cells, a difficult task when you are dealing with an inner ear that is only a few millimeters in size and encased in bone."
Using a technique called subtractive PCR hybridization, the researchers first amplified the cells' differences and subtracted out what they had in common. This left a handful of genes found only in outer hair cells. The researchers then made an informed choice as to which gene to test first, and, fortunately, the first gene turned out to be the right one.
The researchers next transfected the selected gene into human kidney cells, cells which ordinarily do not exhibit microscopic movement. The gene's prestin protein was expressed in the cells, and, when the researchers applied a voltage, they detected voltage-regulated movement in the kidney cells. This provided functional proof that they had identified the motor gene and protein. In recognition of the motor's speed, the team named the gene Prestin after the musical notation presto, meaning very fast tempo.
The speed of the motor, the researchers said, and the fact that it does not require extra biological energy but only a voltage, such as that from a battery, to function, holds promise for applications in nanotechnology.
"Here we have a molecule that directly converts electricity into mechanical force," said Dallos. "This novel motor potentially could be used to build machines on the molecular scale." The other biological motors are poor candidates because they are slow and do require additional energy to work.
"This is an exciting development for nanotechnology," said Laurence Marks, professor of materials science and engineering at Northwestern and an expert in nanotechnology, electron microscopy and atomic structures. "One can envisage creating hybrid structures using this protein in artificial membranes that are part of Nano Electro-Mechanical Systems (NEMS), for instance biological pumps that might be used for drug delivery."
Dallos and Madison, however, probably will leave the nanotechnology applications to other researchers. With the Prestin gene in hand, the researchers have plotted their next steps in learning more about prestin and about hearing. Their primary goal is to create animals that lack the prestin protein and then study how these animals hear. For example, how does the absence of the motor impact hearing? They also want to better characterize the structure of the protein and understand how the molecule produces displacements at such phenomenal speeds. And, with better understanding of the motor protein, the researchers hope to conclusively show how the amplification process of the outer hair cells works.
"For more than 30 years, Peter Dallos has been at the forefront worldwide in the research of the peripheral auditory system," said David Hanson, M.D., chair of otolaryngology at the Medical School and director of the Center for Sensory and Communication Disorders at Northwestern. "The identification of Prestin is a very important contribution to understanding the genetics of hearing disorders, an area in which we are making great strides."
In addition to Dallos and Madison, other authors on the paper are first author Jing Zheng, David Z.Z. He and Weixing Shen from Northwestern University's Auditory Physiology Laboratory and Kevin B. Long from the University's Medical School.
The research was supported by a Senior Fellowship to Peter Dallos from the McKnight Endowment Fund for Neuroscience and the National Institute on Deafness and Other Communication Disorders.
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