Date of Award

1-12-2007

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Chun Jiang - Chair

Second Advisor

Deborah Baro

Third Advisor

Charles Derby

Fourth Advisor

Phang C. Tai

Abstract

Ligand-gated ion channels couple intra- and extracellular chemical signals to cellular excitability. In response to a specific ligand, these channels change their permeability to certain ions by opening or closing their ion conductive pathway, a controlling mechanism known as channel gating. Although recent studies with X-ray crystallography and site-directed mutagenesis have revealed several structures potentially important for channel gating, the gating mechanism is still elusive. Ligand-dependent channel gating involves a series of transient events and asymmetric movements of individual subunits. Understanding of these events appears to be a challenge to current approaches in gating studies by using the homomeric wild-type or mutant channels. I therefore took an alternative approach by constructing heteromeric channels. Subunit stoichiometric studies of the Kir1.1 channel showed that a minimum of one functional subunit was required for the pH-dependent gating of the channel. Four subunits in this channel were coordinated as dynamic functional dimers. In Kir6.2 channel, stoichiometry for proton-binding was almost identical to that for channel gating in the M2 helix, suggesting a one-to-one direct coupling of proton binding in C-terminus to channel gating in M2 helix. Positive cooperativity was suggested among subunits in both the proton binding and channel gating. Ligand binding can be differentiated from channel gating by studying the ATP-dependent gating of Kir6.2 channel. Disruptions in ATP binding were found to change both the potency and efficacy of the concentration-dependent curves, while the baseline activity instead of maximum inhibition was affected by disruptions of channel gating. Four subunits in the Kir6.2 channel undergo negative cooperativity in ATP binding and positive cooperativity in channel gating. The ligand binding was coupled to the gating mechanism in the same subunit and neighboring subunits, although the intrasubunit coupling was more effective. These results are well described with the operational model which we have applied to ion channel studies for the first time. By manipulating the relative distance and the interaction of two transmembrane helices, the inner helix bundle of crossing was found to not only serve as a gate but also determine the consequence of ligand binding.

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