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Neuroscience Graduate Program at UCSF
Molecular Studies of Potassium Channels
Potassium channels are widely distributed. In the brain, potassium channels regulate neuronal signaling. Potassium channels may also regulate cell volume and the flow of salt across epithelia; control heart rate, vascular tone, and the release of hormones such as insulin; and protect neurons and muscles under metabolic stress.
How can potassium channels serve so many different physiological functions? Potassium channels come in many different flavors; they differ in how their activities are regulated as well as the exact manner they allow passage of potassium ions. Many different potassium channels often coexist in a cell. This richness in potassium channel variety is one of the factors that stemmed early attempts for biochemical purification of potassium channels.
How does a potassium channel allow potassium ions but not the smaller sodium ions to go through? How does a potassium channel alter its activity in response to electrical and chemical signals? How do potassium channels contribute to signaling and plasticity in the brain? How does a cell control the number and type of potassium channels in its subcellular compartments? How might potassium channels have arisen during evolution? We have been fascinated with these questions and believe that what potassium channels will teach us may also be relevant to other membrane proteins.
To study potassium channels, we have chosen a molecular approach that isolates individual potassium channel genes so that the channels they give rise to can be studied one at a time and then compared with potassium channels in native tissues. This molecular study was initiated by positional cloning of the Shaker voltage-gated potassium (Kv) channel gene in the fruit fly and expression cloning of mammalian inwardly rectifying potassium (Kir) channels, founding members of two large, distantly related families of potassium channels in organisms ranging from bacteria to humans.
Potassium channel mutations cause diseases of the brain (epilepsy, episodic ataxia), ear (deafness), heart (arrhythmia), muscle (myokymia, periodic paralysis), kidney (hypertension), pancreas (hyperinsulinemic hypoglycemia, neonatal diabetes), and developmental abnormalities of neural crest–derived tissues (Andersen's syndrome). Conversely, the KCNK9 potassium channel gene acts as a dominant oncogene and is amplified or otherwise overexpressed in several types of human carcinomas. Because of the critical physiological functions of potassium channels, potassium channel openers and blockers have been developed for pharmaceutical purposes. A better understanding of potassium channel function will not only satisfy our curiosity but also have clinical significance.
How do we study potassium channels? One unique advantage in channel studies is the possibility to examine one channel at a time, with submillisecond resolution, for many seconds, in experimentally determined intracellular and extracellular environments. In addition to conducting biophysical, biochemical, and cell biological studies of channel assembly, trafficking, regulation, and function, we need to learn how potassium channels are targeted to specific subcellular compartments of neurons in the mammalian brain, and how they respond dynamically to neuronal activity and in turn modulate neuronal signaling. To understand how potassium channels work, we must explore advances in genomics as well as genetics, and incorporate any useful methodologies suited for membrane proteins.Potassium Channel Functions Probed by Yeast Screens of Randomly Mutagenized Mammalian Kir Channels and Plant Kv Channel
To complement structure-function studies based on site-directed mutagenesis, we took advantage of the ability of certain animal and plant potassium channels to rescue potassium-transport-deficient yeast for growth in low-potassium medium, and screened many hundreds of thousands of randomly mutagenized channels. Such unbiased mutant screens have been instructive. (These studies were partially supported by a grant from the National Institute of Mental Health.)
For the Kir channels with two transmembrane segments per alpha subunit, we deduced a helix-packing model for the Kir2.1 channel distinct from that based on the KcsA structure. We verified our model by isolating second-site suppressors on the side of the M1 membrane-spanning helix facing the lethal mutations they suppress on the M2 membrane-spanning helix, and vice versa. In sequence minimization experiments, we showed that 40 M1 residues predicted to face lipid can all be substituted with the same hydrophobic amino acid, regardless of its size, without losing channel function. Moreover, simultaneous substitution of 16 M2 residues predicted to line the pore with the negatively charged aspartate residues also results in functional channels. Remarkably, these 40 lipid-facing and 16 pore-lining residues are situated, as predicted by our model, in the KirBac1.1 channel structure subsequently solved by Declan Doyle's laboratory (University of Oxford), and—as to be expected— all positions intolerant of substitution are buried within the channel protein.
For the Kv channels with six transmembrane segments per alpha subunit, we employed a similar strategy to deduce a helix-packing model for the plant channel KAT1 at the "down" state adopted by the channel when the electrical potential on the intracellular side of the membrane is much more negative than that outside the cell (i.e., when the membrane potential is hyperpolarized). We have experimentally confirmed predicted interactions between the voltage sensor and the pore domain, thus verifying our model for the channel at the down state.
The unbiased yeast screens have also provided insights on features that enable potassium channels to allow potassium but not the slightly smaller sodium ions to go through (known as potassium selectivity). Starting with random mutagenesis of a mutant Kir3.2 (GIRK2) channel that is constitutively active but allows sodium as well as potassium to go through its pore, thereby compromising yeast growth, we have isolated channels that have "evolved" to become selective for potassium permeation and hence can support yeast growth. The surprising finding that potassium selectivity can be restored by electrostatic stabilization of ions, in a region of the pore (the channel cavity) that is much wider than the selectivity filter, can be accounted for in a kinetic model for the long pore of a potassium channel that harbors multiple ions.
Axonal Targeting and Local Translation of Kv1 Channels in Hippocampal Neurons
Axonal Kv1 channels in the Shaker family enable action potential propagation to the nerve terminals to trigger transmitter release without the undesirable effects of back propagation along the axon; such hyperexcitability due to altered Kv1 channel activity accounts for some of the symptoms of patients with episodic ataxia type 1 and the shaking phenotype of Shaker mutant flies. The number and placement of Kv1 channels along the axon also play important roles in controlling the extent of action potential invasion into the branches of axons. To understand the regulation of axonal Kv1 channels, we first identified the axonal targeting machinery, and found to our surprise that the microtubule plus end–binding protein EB1 as well as the KIF3 kinesin motor are required for Kv1 channel axonal targeting.
Although Kv1 channels primarily reside in axons, they have also been found in somatodendritic regions of neurons in the brain. We have found Kv1.1 mRNA in the dendrites. Our study further uncovered activity regulation of dendritic Kv1.1 local translation. This regulation corresponds to a positive feedback in which increased excitatory synaptic inputs causing activation of the NMDA glutamate receptors will lead to suppression of dendritic Kv1.1 channels and enhanced excitability. (These studies were partially supported by a grant from the National Institute of Mental Health.)
Current Lab Personnel on Lab Website
Link to Additional Publications
Ma, D., Zerangue, N. Raab-Graham, K., Fried, S.R., Jan, Y.N., and Jan, L.Y. (2002). Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart. Neuron 33:715-729.
Gu, C., Jan, Y.N., and Jan, L.Y. (2003). A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channel. Science 301:646-649.
Lai, H.C., Grabe, M., Jan, Y.N., and Jan, L.Y. (2005). The S4 voltage sensor packs against the pore domain in the KAT1 voltage-gated potassium channel. Neuron 47:395-406.
Huang, C.S., Shi, S.-H., Ule, J., Ruggiu, M., Barker, L.A., Darnell, R.B., Jan, Y.N., and Jan, L.Y. (2005). Common molecular pathways mediate long-term potentiation of synaptic excitation and slow synaptic inhibition. Cell 123:105-118.
Wiser, O., Qian, X., Ehlers, M., Ja, W.W., Roberts, R.W., Reuveny, E., Jan, Y.N., and Jan, L.Y. (2006). Modulation of basal and receptor-induced GIRK potassium channel activity and neuronal excitability by the mammalian PINS homolog LGN. Neuron 50:561-573.
Raab-Graham, K.F., Haddick, P.C., Jan, Y.N., and Jan, L.Y. (2006). Activity- and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites. Science 314:144-148.
Gu, C., Zhou, W., Puthenveedu, M.A., Xu, M., Jan, Y.N., and Jan, L.Y. (2006). The microtubule plus-end tracking protein EB1 is required for Kv1 voltage-gated K+ channel axonal targeting. Neuron 52:803-816.
Grabe, M., Lai, H.C., Jain, M., Jan, Y.N., and Jan, L.Y. (2007). Structure prediction for the down state of a potassium channel voltage sensor. Nature 445:550-553.
Shaw, R., Fay, A., Puthenveedu, M.A., von Zastrow, M., Jan, Y.N., and Jan, L.Y. (2007). Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adheren junctions. Cell 128:547-560.
Lily Jan , Ph.D.
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