Potassium channels (K channels) are critical components of electrical signaling, a basic biological process that is essential to the function of nerve and muscle. The channels themselves are gated pores; K ions flow through the pore when it is opened (by voltage or signaling molecules), producing an ionic current that electrically """"""""relaxes"""""""" the nerve or muscle cell. The voltage- and calcium-activated """"""""maxi-K"""""""" channel has served as a prototype for understanding K channel modulation and gating. In terms of physiology, maxi-K channels are especially important in the relaxation of vascular smooth muscle to regulate blood pressure. Despite our knowledge of the function of these channels, little is known of their structure or of the molecular basis of their function. It is hoped that a better understanding of maxi-K channel structure and function will ultimately lead to advances in the treatment of neurological and cardiovascular disease. The maxi-K channel, like other voltage-dependent K channels, undergoes a series of conformational changes when it gates. In this proposal, we aim to test and expand upon recent hypotheses of these gating movements, using a combination of patch-clamp recording and time-resolved fluorescence spectroscopy. Our patch-clamp and spectroscopy experiments will be performed on channels in intact, living cells, and can thus answer questions that cannot be addressed with crystallography. By detecting specific fluorescence quenching interactions between attached fluorophores and endogenous sidechains in the channel, we can obtain estimates of intermolecular distances, and thus gain insight toward channel structure.
Our specific aims are: 1) to determine secondary structural features of the maxi-K channel by estimating distances between sidechains, using independent but complementary approaches of electrophysiology and fluorescence spectroscopy; 2) to determine the solvent accessibility of specific amino acid positions on the channel, by measuring excited-state lifetimes of fluorescently-labeled channels in the presence of iodide (an aqueous quenching agent); and 3) to locate amino acid positions that sense gating movements, by performing fluorescence spectroscopy on open and closed channels in patch-clamped whole cells. This combination of experiments will contribute to significant advances in our knowledge of K channel gating.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM068523-06
Application #
7636293
Study Section
Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
Program Officer
Shapiro, Bert I
Project Start
2004-04-01
Project End
2009-09-15
Budget Start
2008-07-02
Budget End
2009-09-15
Support Year
6
Fiscal Year
2008
Total Cost
$154,461
Indirect Cost
Name
Temple University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Smith, Frank J; Rothberg, Brad S (2013) Analysis of Ca(2+)-binding sites in the MthK RCK domain by X-ray crystallography. Methods Mol Biol 998:277-87
Abarca-Heidemann, Karin; Duchardt-Ferner, Elke; Woehnert, Jens et al. (2013) Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy. Methods Mol Biol 998:289-300
Smith, Frank J; Pau, Victor P T; Cingolani, Gino et al. (2012) Crystal structure of a Ba(2+)-bound gating ring reveals elementary steps in RCK domain activation. Structure 20:2038-47
Soboloff, Jonathan; Rothberg, Brad S; Madesh, Muniswamy et al. (2012) STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol 13:549-65
Rothberg, Brad S (2012) The BK channel: a vital link between cellular calcium and electrical signaling. Protein Cell 3:883-92
Pau, Victor P T; Smith, Frank J; Taylor, Alexander B et al. (2011) Structure and function of multiple Ca2+-binding sites in a K+ channel regulator of K+ conductance (RCK) domain. Proc Natl Acad Sci U S A 108:17684-9
Pau, Victor P T; Abarca-Heidemann, Karin; Rothberg, Brad S (2010) Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel. J Gen Physiol 135:509-26
Thomson, Andrew S; Rothberg, Brad S (2010) Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel. J Gen Physiol 136:569-79
Semenova, Nina P; Abarca-Heidemann, Karin; Loranc, Eva et al. (2009) Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels. J Biol Chem 284:10684-93
Wang, Bin; Rothberg, Brad S; Brenner, Robert (2009) Mechanism of increased BK channel activation from a channel mutation that causes epilepsy. J Gen Physiol 133:283-94

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