Key protein involved in smell identified

Researchers led by scientists from the Hopkins School of Medicine have isolated the protein directly responsible for the optimal perception of odors in vertebrates.

The results of their study, published this November in Nature Neuroscience, point to the action of NCKX4, a protein located in the membrane of olfactory sensory neurons (OSNs), the cells responsible for interpreting odors in the brain. NCKX4 is a potassium dependent sodium/calcium exchanger. Basically, it channels positively charged ions into and out of the cell. When the OSNs are stimulated, calcium ions enter the cells at their cilia, projections from the main cell body that increase the probability that odors can be detected. The flow of calcium ions positively affects odor perception by depolarizing the membrane. It also has a negative effect by adapting the cell over time to reduce sensitivity to odors.

This class of protein harnesses the energy of a gradient of sodium ions across the membrane to pump calcium ions into the cell against the natural flow of the sodium gradient. For every four sodium ions, one calcium ion is transported (an additional potassium ion is also pumped into the cell).

Previous studies have shown that calcium ions have crucial roles in the activation, termination and adaptation of responses to stimuli perceived through the senses. For example, in the rod and cone cells located in the eye, the concentration of calcium inside the cell regulates sensitivity to backlight perceived by the eye.

Additionally, low levels of sodium ions outside the cell have been shown to improve adaptation to odors, suggesting that the movement of both calcium and sodium ions is responsible for adaptation. This eliminates proteins that only transport calcium across the membrane.

The researchers decided to focus on NCKX4 because it is the only sodium/calcium transporter that is present in high concentrations at the very edge of OSN tissues – the epithelium. To study the effect of the protein, they used normal mice and genetically engineered mice in whose DNA the gene for NCKX4 had been deleted (so-called "knockout" mice).

To measure the effect of the absence of NCKX4 on the cells, they used a technique that measures the strength of the electric field at the surface of the cells. They first exposed the mice to a very brief smell stimulus for 100 ms. Then measuring the electric field strength, they analyzed the OSNs' response to the odor stimulus. Their results showed that there was no change in activation – the peak amplitude, response time and time to peak were similar for both normal and knockout mice. This surprising result was attributed to the opposing effect of calcium ions in the cell – the positive depolarization effect and the negative adaptation response. In normal mice, these two effects negate each other. When the protein is absent these effects do not occur and the response appears the same for both types of mice.

However, there was a notable difference on termination of the signal. In knockout mice, the time taken for termination of the response lengthened considerably, to between double and triple that for normal mice.

To effectively test the adaptation of the response – they used a paired-pulse protocol with two equal 100 ms odor pulses. For normal mice, the response to the second pulse was less than that of the first. But, for the knockout mice, the response to the second pulse was even less than that for the normal mice.

Additional evidence for the important effect of NCKX4 on odor response is a huge decrease in the weight of knockout mice when compared to that of normal mice. This correlation appears because detecting odors is valuable in young mice for suckling – their main source of food as infants. Additionally, the cells in knockout mice fired fewer action potentials, sending fewer signals from the OSNs to the brain in response to odor stimuli.