The long term objectives of this proposal are: to define the structural determinants and mechanisms of calcium and voltage activation of """"""""maxim Kca channels, and to determine the protein domains responsible for their modulation by phosphorylation, G proteins and beta subunits. We have recently cloned and functionally expressed a human maxi Kca channel, hslo, and a splice variant (hslo-A). Preliminary data shows that: i) co- expression of hslo, the pore-forming alpha subunit, with its beta subunit dramatically increases its Ca2+ sensitivity; and 2) hslo can be modulated by Gs proteins. Thus, we will study, by mutagenesis, chimera construction, pharmacology, and site-directed antibodies, the functional coupling between hslo and these modulatory proteins, and the structural determinants of their interaction. The main questions that we want to address are: 1) how can we explain Kca channel functional diversity in native tissues? 2) can we identify the domains responsible for Ca2+- and voltage-dependent activation? 3) how do phosphorylation and G proteins regulate the pore- forming a subunit and what domains are involved? and 4) what are the mechanisms and the domains for the beta subunit action on hslo function? To answer these questions, studies will be performed primarily by using hslo, its beta subunit, cGMP-protein kinase and G protein clones. In order to compare naturally occurring mutations, we will study the two Kca channel clones (hslo and hslo-A) already available, and isolate other hslo splicing variants and human homologue(s) of the beta subunit.
The specific aims are to: 1) clone a human homologue(s) of the hslo alpha subunit and other splice variants of hslo. We will investigate if different splice variants behave equally, and whether hslo can couple with different isoforms of beta subunits. We will also examine the mRNA distribution of a and beta subunits under different hormonal conditions; 2) investigate the mechanism(s) of voltage- and Ca2+-dependent activation. To investigate the coupling of Ca2+ binding, charge movement and pore opening we will measure the calcium and voltage sensitivities of ionic and gating currents in hslo, hslo homologues, and in carboxyl terminus and S4 region mutants; 3) examine the modulation of the pore-forming alpha subunit by phosphorylation (PKA, cGMP- PK) and putative G protein action (Gs-alpha, beta-gamma subunits), and establish the sites of modulation by phosphorylation. We will analyze modifications in the voltage and calcium dependencies induced by these modulatory mechanisms; and 4) study the structural determinants of the interaction between the alpha and beta subunits of the KCa channel complex, and the functional consequences. Structure-function studies of human Kca channels may be useful in the design of therapeutic means to prevent smooth muscle spasm in coronary disease or premature labor.
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