Research in the Nichols Lab

Our research group is focused on the molecular and cellular regulation of potassium channels, and their role in linking cellular metabolism to electrical activity in various tissues. We have developed a detailed understanding of the molecular basis of potassium channel function as well as clinically relevant understanding of the mechanistic basis of inherited potassium channel diseases. Our latest efforts are directed towards a more complete understanding of the molecular details, the physiological role, and the clinical relevance, of potassium channel activity, using combinations of biochemical, genetic, physiological and biophysical approaches.

Current Research Opportunities

Overview

For fifteen years, the broad context of our research has been the molecular basis, and functional role, of potassium channels. The early cloning of channel genes led to understanding the molecular basis of function by mutagenic and now crystallographic methods. In parallel, these basic studies have fuelled the genetic manipulation of channels in recombinant organisms, in turn explaining ion channel disease etiologies and informing clinical research. Our own major contributions include (1) the cloning and expression of novel K channel genes, (2) elucidation of the mechanism of K (Kir) channel rectification, (3) elucidation of the molecular basis of KATP channel function, (4) the first molecular model of the Kir channel pore, (5) transgenic animal models of diabetes, hyperinsulinism, and cardiac dysrrhythmia. Current major challenges are atomic resolution of Kir channel structure and function, and further elucidation of the roles of Kir channels in organ function and dysfunction.

The mechanism of K channel rectification: The structural basis of K channel activity

A major class of K channels are the so-called inward rectifiers (Kir). Classic Kir channels have a very steep voltage-dependence of conductance that permits them to pass inward currents far more easily than outward currents. Since its' discovery over 50 years ago, the phenomenon of inward rectification, remained largely unexplained until we cloned a strong inward rectifier1 and went on to demonstrate that rectification requires soluble, low molecular weight compounds, and identified them as polyamines2,3. This work opened up a renewed interest in the phenomenon of inward rectification. Other groups went on to show that polyamines also underlie inward rectification of many other channel types. We are currently pursuing the detailed biophysics and structural requirements of polyamine induced inward rectification4-10, as well as the physiological and clinical relevance of these findings11,12.
In order to approach the structural basis of channel activity, we performed systematic cysteine-scanning analysis of the K channel pore6,7 as a prelude to eventual crystallization of a Kir channel. Our findings of phospholipid control of KATP channel activity13 suggested a critical lipid-binding domain in the C-terminus of the channel14-16. To move this understanding to the next level, we embarked on ambitious projects with recombinant channel proteins. We have cloned, reconstituted activity of17, and crystallized, a bacterial Kir homolog. These studies lay the basis for combined crystallographic and functional studies that will lead us to the final goal of atomic resolution of Kir channel function.

Molecular mechanism of KATP channel regulation

ATP-sensitive K channels link metabolism to electrical activity in numerous tissue. We have long sought th answer to the question: What is the nature of ATP inhibition of KATP channels? Ultimately, the answer to this question requires knowledge of the protein structure. We first cloned and expressed many inwardly rectifying (Kir) channel genes1,5,17-19, as well as a KATP channel component, the sulfonylurea-binding protein (SUR120). We eventually reconstituted channel activity by coexpression of SUR1 with a Kir channel subunit (Kir6.221-23. Consistent with the KATP channel being intimately involved with the regulation of insulin secretion, Dr. A. Permutt (Division of Endocrinology and Metabolism) and colleagues discovered numerous mutations in the SUR1 gene from patients with defects of insulin secretion (diabetes and persistent hypoglycemic hyperinsulinemia – PHHI24,25. We showed that a mutation from one PHHI patient changes the amino acid sequence in one of the nucleotide binding folds of SUR1, altering nucleotide regulation of the channel in such a way as to cause the PHHI phenotype26. We moved on to systematic structure-function analysis of the channel complex, demonstrating an 8-fold (4 SUR1 + 4 Kir6.2 subunits) stoichiometry of the channel22, and providing detailed insight into the role of Kir6.2 subunit in forming the channel pore21, and of the SUR1 and Kir6.2 subunits in nucleotide regulation23,27,28.
Our latest efforts are directed to quantitative mechanistic explanation of gating. We have developed very sophisticated kinetic models of gating29-31, and allied these with biophysical10,32,33 and biochemical measurements. Cloning and reconstitution17 as well as crystallization of a bacterial K channel (above) now points the way to final elucidation of the molecular basis of KATP channel function.

Pathophysiology of K channels

In addition to further reductionist approaches to understanding Kir and KATP channel function, we are well placed to examine the physiological function of channel genes in transgenic animals. We used a genetically mutated cell line deficient in ornithine decarboxylase activity to manipulate polyamine levels and demonstrate the role of polyamines in regulating inward rectification11. We subsequently manipulated inward rectifier K channels in transgenic animals expressing altered polyamine synthetic enzyme levels12, leading to the generation of novel transgenic animals with altered Kir channels to probe their role in cardiac function that are now being examined by our colleague Dr. Lopatin at the University of Michigan.
Using green-fluorescent-protein (GFP) tagged constructs, we opened a window to the cell biology of the KATP channel complex, early studies demonstrating independent trafficking of the two subunits34. We have gone on to utilize GFP-tagged channel constructs to generate transgenic mice expressing mutant KATP channel activity in pancreatic and cardiac cells. We demonstrated hyperinsulinism in animals expressing reduced KATP channel activity[Koster, 2002 4552]35 and a dramatic diabetic phenotype as a consequence of KATP channel overactivity in ?-cells36. This latter work predicted a correlate human disease, and recent work from Andrew Hattersley and colleagues has shown that similar gain-of-functionmutations in KATP indeed underlie human neonatal diabetes.
By contrast, the heart shows a remarkable tolerance for reduced sensitivity of KATP channel activity37,38, a feature that we are now examining the underlying mechanism of39,40. We are currently developing novel inducible transgenic strategies, as well as utilizing alternative viral-mediated approaches, which promise insight to mechanisms of ischemic protection and preconditioning, as well as to membrane lipid regulation of cardiac and pancreatic function.

Selected references

1.Makhina, E.N., A.J. Kelly, A.N. Lopatin, R.W. Mercer, et al., Cloning and expression of a novel human brain inward rectifier potassium channel. Journal of Biological Chemistry, 1994. 269: 20468-74.
2.Lopatin, A.N., E.N. Makhina, and C.G. Nichols, Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature, 1994. 372: 366-9.
3.Lopatin, A.N., E.N. Makhina, and C.G. Nichols, The Mechanism Of Inward Rectification Of Potassium Channels - Long-Pore Plugging By Cytoplasmic Polyamines. Journal of General Physiology, 1995. 106: 923-955.
4.Lopatin, A.N. and C.G. Nichols, [K+] dependence of polyamine-induced rectification in inward rectifier potassium channels (IRK1, Kir2.1). Journal of General Physiology, 1996. 108: 105-13.
5.Pearson, W.L. and C.G. Nichols, Block of the Kir2.1 channel pore by alkylamine analogues of endogenous polyamines. J Gen Physiol, 1998. 112: 351-63.
6.Loussouarn, G., E.N. Makhina, T. Rose, and C.G. Nichols, Structure and dynamics of the pore of inwardly rectifying K(ATP) channels. J Biol Chem, 2000. 275: 1137-44.
7.Loussouarn, G., L.R. Phillips, R. Masia, T. Rose, et al., Flexibility of the Kir6.2 inward rectifier K(+) channel pore. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98: 4227-32.
8.Loussouarn, G., T. Rose, and C.G. Nichols, Structural basis of inward rectifying potassium channel gating. Trends in Cardiovascular Medicine, 2002. 12: 253-8.
9.Loussouarn, G., L.J. Marton, and C.G. Nichols, Molecular basis of inward rectification: structural features of the blocker defined by extended polyamine analogs. Molecular Pharmacology, 2005. 68: 298-304.
10.Kurata, H.T., L.R. Phillips, T. Rose, G. Loussouarn, et al., Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis. Journal of General Physiology, 2004. 124: 541-554.
11.Shyng, S.L., Q. Sha, T. Ferrigni, A.N. Lopatin, et al., Depletion of intracellular polyamines relieves inward rectification of potassium channels. Proceedings of the National Academy of Sciences of the United States of America, 1996. 93: 12014-9.
12.Lopatin, A.N., L.M. Shantz, C.A. Mackintosh, C.G. Nichols, et al., Modulation of potassium channels in the hearts of transgenic and mutant mice with altered polyamine biosynthesis. J Mol Cell Cardiol, 2000. 32: 2007-24.
13.Shyng, S.L. and C.G. Nichols, Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science, 1998. 282: 1138-41.
14.Shyng, S.L., C.A. Cukras, J. Harwood, and C.G. Nichols, Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels [In Process Citation]. J Gen Physiol, 2000. 116: 599-608.
15.Cukras, C.A., I. Jeliazkova, and C.G. Nichols, Structural and functional determinants of conserved lipid interaction domains of inward rectifying kir6.2 channels. J Gen Physiol, 2002. 119: 581-91.
16.Cukras, C.A., I. Jeliazkova, and C.G. Nichols, The role of NH(2)-terminal positive charges in the activity of inward rectifier K(ATP) channels. J Gen Physiol, 2002. 120: 437-46.
17.Enkvetchakul, D., J. Bhattacharyya, I. Jeliazkova, D.K. Groesbeck, et al., Functional characterization of a prokaryotic Kir channel. Journal of Biological Chemistry, 2004. 279: 47076-80.
18.Ho, K., C.G. Nichols, W.J. Lederer, J. Lytton, et al., Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature, 1993. 362: 31-8.
19.Ferrer, J., C.G. Nichols, E.N. Makhina, L. Salkoff, et al., Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels. Journal of Biological Chemistry, 1995. 270: 26086-91.
20.Aguilar-Bryan, L., C.G. Nichols, S.W. Wechsler, J.P.t. Clement, et al., Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science, 1995. 268: 423-6.
21.Shyng, S., T. Ferrigni, and C.G. Nichols, Control of rectification and gating of cloned KATP channels by the Kir6.2 subunit. Journal of General Physiology, 1997. 110: 141-53.
22.Shyng, S. and C.G. Nichols, Octameric stoichiometry of the KATP channel complex. J Gen Physiol, 1997. 110: 655-64.
23.Shyng, S., T. Ferrigni, and C.G. Nichols, Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor. J Gen Physiol, 1997. 110: 643-54.
24.Shyng, S.L., T. Ferrigni, J.B. Shepard, A. Nestorowicz, et al., Functional analyses of novel mutations in the sulfonylurea receptor 1 associated with persistent hyperinsulinemic hypoglycemia of infancy. Diabetes, 1998. 47: 1145-51.
25.Nestorowicz, A., B. Glaser, B.A. Wilson, S.L. Shyng, et al., Genetic heterogeneity in familial hyperinsulinism [published erratum appears in Hum Mol Genet 1998 Sep;7(9):1527]. Hum Mol Genet, 1998. 7: 1119-28.
26.Nichols, C.G., S.L. Shyng, A. Nestorowicz, B. Glaser, et al., Adenosine Diphosphate As an Intracellular Regulator Of Insulin Secretion. Science, 1996. 272: 1785-1787.
27.Koster, J.C., Q. Sha, and C.G. Nichols, Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits. Journal of General Physiology, 1999. 114: 203-13.
28.Koster, J.C., Q. Sha, S. Shyng, and C.G. Nichols, ATP inhibition of KATP channels: control of nucleotide sensitivity by the N-terminal domain of the Kir6.2 subunit. Journal of Physiology, 1999. 515: 19-30.
29.Enkvetchakul, D., G. Loussouarn, E. Makhina, and C.G. Nichols, ATP interaction with the open state of the K(ATP) channel. Biophysical Journal, 2001. 80: 719-28.
30.Enkvetchakul, D., G. Loussouarn, E. Makhina, S.L. Shyng, et al., The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding. Biophys J, 2000. 78: 2334-48.
31.Enkvetchakul, D. and C.G. Nichols, Gating mechanism of KATP channels: function fits form. Journal of General Physiology, 2003. 122: 471-80.
32.Phillips, L.R., D. Enkvetchakul, and C.G. Nichols, Gating dependence of inner pore access in inward rectifier K(+) channels. Neuron, 2003. 37: 953-62.
33.Phillips, L.R. and C.G. Nichols, Ligand-induced closure of inward rectifier Kir6.2 channels traps spermine in the pore. Journal of General Physiology, 2003. 122: 795-804.
34.Makhina, E.N. and C.G. Nichols, Independent trafficking of KATP channel subunits to the plasma membrane. Journal of Biological Chemistry, 1998. 273: 3369-74.
35.Remedi, M.-S., J.C. Koster, K.P. Markova, S. Seino, et al., Diet-induced glucose intolerance in mice with decreased b-cell KATP channels. Diabetes, 2004. 53: 3159-3167.
36.Koster, J.C., B.A. Marshall, N. Ensor, J.A. Corbett, et al., Targeted overactivity of beta cell K(ATP) channels induces profound neonatal diabetes. Cell, 2000. 100: 645-54.
37.Koster, J.C., A. Knopp, T.P. Flagg, K.P. Markova, et al., Tolerance for ATP-Insensitive KATP Channels in Transgenic Mice. Circulation Research, 2001. 89: In Press.
38.Rajashree, R., J.C. Koster, K.P. Markova, C.G. Nichols, et al., Contractility and ischemic response of hearts from transgenic mice with altered sarcolemmal K(ATP) channels. American Journal of Physiology - Heart & Circulatory Physiology, 2002. 283: H584-90.
39.Flagg, T.P., F. Charpentier, J. Manning-Fox, M.S. Remedi, et al., Remodeling of excitation-contraction coupling in transgenic mice expressing ATP-insensitive sarcolemmal KATP channels. American Journal of Physiology - Heart & Circulatory Physiology, 2004. 286: H1361-9.
40.Flagg, T.P., M.S. Remedi, R. Masia, M. McLerie, et al., Transgenic overexpression of SUR1 in the heart exerts dominant negative effects on sarcolemmal KATP. Journal of Molecular & Cellular Cardiology, 2005. 39: 647-656.