Richard Kramer

Professor (Molecular & Cell Biology)

Email: rhkramer@berkeley.edu

Research areas: Cellular and Molecular Neuroscience, Cellular and Molecular Neuroscience

Research Interests

Nerve cells communicate using electrical and chemical signals. We use a combination of molecular, optical, and electrophysiological methods to study ion channels, the proteins that generate electrical signals, and synaptic transmission, the process that allows a neuron to communicate chemically with other cells. Many of our most recent studies utilize novel chemical reagents to modify the function of ion channels and synapses. This Chemical-Biological approach is designed to allow non-invasive optical sensing and optical manipulation of neuronal activity in intact regions of the nervous system.

Current Projects

Constructing "light-activated" ion channels for remote control of neural activity. Neurons have ion channels that are activated directly by voltage, chemicals, and mechanical forces, and temperature, but not by light. Using a combination of organic chemistry and molecular biology, we have engineered the first neuronal ion channel that can be directly activated with light. A chemically synthesized "photoswitch" molecule is covalently coupled to genetically engineered ion channel protein. The photoswitch contains a ligand that binds to the pore of the channel, blocking the flow of ions. When the photoswitch is in its extended (trans) configuration, the blocker can reach the pore and the channel remains closed, but exposure to ultraviolet light triggers photoisomerization to the bent (cis) form of the molecule, retracting the blocker from the pore, and allowing ion flow. The photoswitch can be switched rapidly and repeatedly with different wavelengths of light, allowing precise and consistent control of channel opening and closing. Expression of these channels in neurons allows action potentials to be regulated with flashes of light. Light-activated channels could serve as "remote control" devices for non-invasive control of neural activity---an alternative to neural prosthetic devices based on implanted electrode arrays. One particularly intriguing target for these channels is in neurons of the retina---the one part of the nervous system that is naturally accessible to light. By converting "blind" retinal neurons (e.g. retinal ganglion cells) into artificially photosensitive cells, it may be possible to restore visual sensitivity to blind animals that have lost their natural photoreceptors (rods and cones) to injury or degenerative diseases.

Optical studies of synaptic transmission in the retina. Rod and cone photoreceptors of the retina transmit information to other neurons through specialized structures called ribbon synapses. Unlike most synapses, ribbon synapses continuously release neurotransmitter, with the sensory stimulus (light) causing a decrease in the rate of release. We are using fluorescent indicator dyes and proteins, along with 2-photon, confocal, and electron microscopy, to track the life cycle of synaptic vesicles in photoreceptor terminals. This process starts with endocytosis, includes interactions with the synaptic ribbon, and ends with exocytosis. We are also examining how physiological stimuli, including light and synaptic feedback from other retinal neurons, regulate exocytosis from rods and cones. The use of activity-dependent dyes allows us to examine not only individual synapses, but also the behavior of 2-dimensional arrays of synapses while we project visual images on the retina.

Patch cramming: monitoring intracellular cGMP in intact neurons. We have developed a simple method for monitoring cGMP in intact cells. We use a micropipette to obtain a membrane patch from a Xenopus oocyte expressing cyclic nucleotide-gated channels that have been engineered to be sensitive and selective for cGMP. After calibrating its cGMP response, the pipette is "crammed" into a sufficiently large heterologous cell. Once inside, the activity of CNG channels in the patch reflects the cytoplasmic cGMP concentration. Suitable recipient cells include retinal horizontal cells, neuroblastoma cells, dorsal root ganglion cells, and cerebellar Purkinje cells. Using patch cramming we are obtaining the first glimpses of cGMP dynamics in response to neurotransmitters and other stimuli. In particular, we find that some transmitters trigger long-term suppression of cGMP, which is potentially important for synaptic plasticity.

Modulation of phototransduction by growth factors and tyrosine phosphorylation. The light sensitivity of rods and cones decreases with background illumination---a process known as light adaptation. Light adaptation allows our visual system to adjusts ambient light levels of during night and day, allowing us to see clearly in a wide range of conditions. Rapid processes of light adaptation that are intrinsic to rods and cones have been studied for many years. We are investigating a slower, unexplored type of adaptation that is mediated by an extrinsic signal, namely the release of growth factors onto rods by neighboring pigment epithelial cells. Growth factor signal transduction often involves protein kinases and phosphatases specific for tyrosine residues. We find that insulin-like growth factor (IGF-1) induces changes in tyrosine phosphorylation, increasing the sensitivity of rod CNG channels and thus altering the light response. Ongoing studies are aimed at elucidating the biochemical cascade linking the IGF-1 receptor and the CNG channel and understanding how tyrosine phosphorylation triggers conformational changes that alter channel activity. Additional studies on transgenic animals will determine the consequences of growth factor effects on vision.

Selected Publications

A. Savchenko, T.W. Kraft, E. Molokanova, and R. H. Kramer 2001. Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through phosphorylation of a specific tyrosine residue Proc. Natn. Acad. Sci. USA 98: 5880-5885.

Kramer, R. H. and Karpen, J. 1998. Spanning binding sites on allosteric proteins with polymer-linked ligand dimers Nature 395: 710-713.

Trivedi, B. & Kramer, R. H. 1998. Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists Neuron 21: 895-906.

Savchenko, A., Barnes, S. A., & Kramer, R. H. 1997. Cyclic nucleotide-gated channels mediate synaptic feedback by nitric oxide Nature 390: 694-698.

E. Molokanova, and R.H. Kramer 2001. Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein J. Gen. Physiol. 117: 219-234.

R.H. Kramer and E. Molokanova 2001. Modulation of cyclic nucleotide-gated channels and regulation of phototransduction J. Exp. Biol. 204: 2921-2931.

B. Trivedi, and R.H. Kramer 2002. Patch cramming reveals the mechanism of ling-term suppression of cyclic nucleotides in intact neurons J. Neurosci. 22: 8819-8826.

Choi, S.Y., Rea, R., Borghuis, B, Sterling, P., and Kramer, R.H. (in press). Synaptic coding of intensity at the cone photoreceptor synapse Neuron.

Banghart, M., Borges, K., Isacoff, E., Trauner, D., and Kramer, R.H. 2004. Light-activated ion channels for remote control of neuronal firing Nature Neuroscience 12: 1381-1386.

Rea, R., Li., J., Dharia, A., Levitan, E.S., Sterling, P., and Kramer, R.H. 2004. Streamlined synaptic vesicle cycle in cone photoreceptor terminals Neuron 41: 755-766.

Krajewski, J.L, Luetje, C.W., and Kramer, R.H. 2003. Tyrosine phosphorylation switches off Ca2+/calmodulin inhibition in rod cyclic nucleotide-gated channels Journal of Neuroscience 23: 10100-10106.

Molokanova, E., Krajewski, JL., Satpaev, D, Luetje, CW, and Kramer, RH 2003. Subunit contributions to phosphorylation–dependent modulation of rod cyclic nucleotide-gated channels Journal of Physiology 552: 345-356.