Research Summary

Potassium Channel Functions Probed by Yeast Screens
of Randomly Mutagenized Mammalian Kir Channels and Plant Kv Channels

Cell Biological Regulation of Potassium Channel Assembly and Trafficking

Publications
         Transmitter actions and synapses
         Voltage-gated potassium channels
         Inwardly rectifying potassium channels
         Trafficking channels and receptors
         Channel families-evolution
         Novel fluorescent probes
         Potassium channels and disease

Contact Lily Jan

Research Summary

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 Channels

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.

Model of Kir channels: Large-scale mutant screens in yeast yield reliable structural information. Model of Kir channels based on the KirBac1.1 structure, showing 10 M1 residues per subunit predicted to face lipid (yellow), 4 M2 residues predicted to line the pore (cyan), and 11 M1 and M2 residues predicted to be buried within the channel protein (red), based on analyses of IRK1 (Kir2.1) mutant channels that rescue potassium-transport-deficient yeast for growth in low-potassium medium. The outer pair (pink and blue) and inner pair (orange and brown) yielding GIRK2 (Kir3.2) gating mutants isolated from unbiased yeast screens are shown on the green subunit on the left.

The amino acids in IRK1 (and corresponding residues given in parentheses for KirBac1.1) are I87(S66), L90(A69), A91(L70), L94(V73), L97(T76), F98(L77), C101(L80), W104(Q83), L105(L84), and L108(A87) for lipid-facing; S165(I131), C169(M135), D172(I138), and I176(T142) for pore-lining; and F92(F71), S95(N74), W96(N75), F99(F78), G100(A79), A107(D86), A157(A123), V158(H124), V161(A127), Q164(E130), and G168(G134) for buried residues. The outer and inner pair of GIRK2 residues important for holding the channel in the closed conformation (and corresponding residues given in parentheses for KirBac1.1) are E152(L108) in pink and S177(I131) in blue; N94(F63) in orange and V188(T142) in brown.

From Minor, D.L., Jr., Masseling, S.J., Jan, Y.N., and Jan, L.Y. 1999. Cell 96:879-891; Yi, B.A., Lin, Y.F., Jan, Y.N., and Jan, L.Y. 2001. Neuron 29:657-667; and Kuo, A., Gulbis, J.M., Antcliff, J.F., Rahman, T., Lowe, E.D., Zimmer, J., Cuthbertson, J., Ashcroft, F.M., Ezaki, T., and Doyle, D.A. (2003) www.sciencemag.org/ 8 May 2003 [DOI:/10.1126/science.1085028].

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.)