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MECHANISM OF CALCIUM DEPENDENT GATING OF BKCa CHANNELS: RELATING PROTEIN STRUCTURE TO FUNCTION

Krishnamoorthy, Gayathri
http://rave.ohiolink.edu/etdc/view?acc_num=case1144444855

Abstract Details

2006, Doctor of Philosophy, Case Western Reserve University, Biomedical Engineering.
Large-conductance voltage and calcium activated K+ (BKCa) channels play regulatory roles in processes involving high intracellular calcium concentrations such as neurotransmitter release, vasoregulation and hearing. BKCa channels rapidly hyperpolarize membrane potentials and terminate calcium influx via voltage-gated calcium channels. Similar to voltage-gated K+ channels, the alpha-subunit of BKCa channel contains a voltage-sensing module, a K+ selective pore and an activation gate composed by N-terminal transmembrane segments. In addition, BKCa has a long intracellular carboxyl terminal containing calcium binding sites and RCK domains that regulate calcium sensitivity of BKCa activation. BKCa activation is an allosteric process wherein the energy gained from calcium and voltage is transmitted to the gate via conformational changes in structures linking calcium binding sites and voltage sensors to the gate. The molecular mechanism of calcium dependent activation is derived from the crystal structure of an archeon calcium activated K+ channel, MthK. In this model, calcium binds to an intracellular gating ring structure, composed of eight RCK domains, leading to conformational changes in the gating ring that pulls the activation gate open via a linker. Here, we explored the allosteric mechanism of calcium dependent activation by comparing two homologous BKCa channels from mouse (mSlo1) and Drosophila melanogaster (dSlo1) that have different calcium sensitivities. Using site-directed mutagenesis, patch-clamp technique, MWC models and molecular dynamic simulations, we identified an allosteric regulatory domain in the N-terminus of the RCK1 domain (AC region) that modulates calcium sensitivity based on calcium occupancy and channel state. Our observations are consistent with a mechanism where the AC region inhibits channel activation when the channel is closed in the absence of calcium; calcium binding and depolarization relieves this inhibition. In a subsequent study, we found that a recently identified epilepsy mutation D369G in the AC region of mSlo1 BKCa alpha-subunit enhanced calcium sensitivity of activation at intermediate [Ca2+]i by modifying the conformation and dynamics of the AC region in an allosteric manner. Further investigation revealed a putative structural network that may form the basis of allosteric coupling of calcium binding sites and the gate by the AC region in calcium dependent BKCa activation.
Jianmin Cui (Advisor)
145 p.
BK channel; Large conductance calcium activated potassium channel; allosteric mechanism; epilepsy; MWC model

Recommended Citations

Citations

  • Krishnamoorthy, G. (2006). MECHANISM OF CALCIUM DEPENDENT GATING OF BKCa CHANNELS: RELATING PROTEIN STRUCTURE TO FUNCTION [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1144444855

    APA Style (7th edition)

  • Krishnamoorthy, Gayathri. MECHANISM OF CALCIUM DEPENDENT GATING OF BKCa CHANNELS: RELATING PROTEIN STRUCTURE TO FUNCTION. 2006. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1144444855.

    MLA Style (8th edition)

  • Krishnamoorthy, Gayathri. "MECHANISM OF CALCIUM DEPENDENT GATING OF BKCa CHANNELS: RELATING PROTEIN STRUCTURE TO FUNCTION." Doctoral dissertation, Case Western Reserve University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=case1144444855

    Chicago Manual of Style (17th edition)

case1144444855
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© 2006, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.