The mapping of functional genes plays an important role in studies of genome structure, function, and evolution, as well as allowing gene cloning and marker-assisted selection to improve agriculturally important traits. Simple sequence repeats (SSRs) developed from expressed sequence tags (ESTs), EST-SSR (eSSR), can be employed as putative functional marker loci to easily tag corresponding functional genes. In this paper, 2218 eSSRs, 1554 from G. raimondii-derived and 754 from G. hirsutum-derived ESTs, were developed and used to screen polymorphisms to enhance our backbone genetic map in allotetraploid cotton. Of the 1554 G. raimondii-derived eSSRs, 744 eSSRs were able to successfully amplify polymorphisms between our two mapping parents, TM-1 and Hai7124, presenting a polymorphic rate of 47.9%. However, only a 23.9% (159/754) polymorphic rate was produced from G. hirsutum-derived eSSRs. No relationship was observed between the level of polymorphism, motif type, and tissue origin, but the polymorphism appeared to be correlated with repeat type. After integrating these new eSSRs, our enhanced genetic map consists of 1790 loci in 26 linkage groups and covers 3425.8 cM with an average intermarker distance of 1.91 cM. This microsatellite-based, gene-rich linkage map contains 71.96% functional marker loci, of which 87.11% are eSSR loci. There were 132 duplicated loci bridging 13 homeologous At/Dt chromosome pairs. Two reciprocal translocations after polyploidization between A2 and A3, and between A4 and A5, chromosomes were further confirmed. A functional analysis of 975 ESTs producing 1122 eSSR loci tagged in the map revealed that 60% had clear BLASTX hits (,1e À10 ) to the Uniprot database and that 475 were associated mainly with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function; many of the ESTs were associated with two or more category functions. The results presented here will provide new insights for future investigations of functional and evolutionary genomics, especially those associated with cotton fiber improvement.

Ion channels are proteins that control the flux of ions across cell membranes by opening and closing (gating) their pores. It has been proposed that channels gated by internal agonists have an intracellular gating ring that extracts free energy from agonist binding to open the gates using linkers that directly connect the gating ring to the gates. Here we find for a voltage- and Ca(2+)-activated K+ (BK) channel that shortening the linkers increases channel activity and lengthening the linkers decreases channel activity, both in the presence and absence of intracellular Ca2+. These observations are consistent with a mechanical model in which the linker-gating ring complex forms a passive spring that applies force to the gates in the absence of Ca2+ to modulate the voltage-dependent gating. Adding Ca2+ then changes the force to further activate the channel. Both the passive and Ca(2+)-induced forces contribute to the gating of the channel.

Large-conductance Ca 2؉ -voltage-activated K ؉ channels (BK channels) control many key physiological processes, such as neurotransmitter release and muscle contraction. A signature feature of BK channels is that they have the largest single channel conductance of all K ؉ channels. Here we examine the mechanism of this large conductance. Comparison of the sequence of BK channels to lower-conductance K ؉ channels and to a crystallized bacterial K ؉ channel (MthK) revealed that BK channels have a ring of eight negatively charged glutamate residues at the entrance to the intracellular vestibule. This ring of charge, which is absent in lower-conductance K ؉ channels, is shown to double the conductance of BK channels for outward currents by increasing the concentration of K ؉ in the vestibule through an electrostatic mechanism. Removing the ring of charge converts BK channels to inwardly rectifying channels. Thus, a simple electrostatic mechanism contributes to the large conductance of BK channels.L arge-conductance Ca 2ϩ -voltage-activated K ϩ (BK) channels are activated in a highly synergistic manner by intracellular Ca 2ϩ (Ca i 2ϩ ) and membrane depolarization (1)(2)(3)(4)(5)(6)(7)(8)(9). When open, the efflux of K ϩ out of the cell hyperpolarizes the membrane potential, turning off voltage-dependent Ca 2ϩ channels and reducing the influx of Ca 2ϩ available to both activate BK channels and control cellular processes. This negative feedback mechanism allows BK channels to play a key role in regulating many physiological processes, such as neurotransmitter release (10, 11), repetitive firing of neurons (12), spike broadening during repetitive firing (13), the electrical tuning of hair cells in the cochlea (14,15), and muscle contraction (16).BK channels (for big K ϩ ) have the largest single-channel conductance of all K ϩ selective channels, being 250-300 pS in symmetrical 150 mM KCl (8,[17][18][19][20]. Like most K ϩ channels of lower conductance, BK channels have a tetrameric structure, with four ␣ subunits forming functional channels. BK channels also have the same selectivity filter sequence (GYG) found in most other K ϩ channels of lower conductance (21). Thus, the question arises as to why the conductance of BK channels is so big.Previous experimental and theoretical work has suggested that charged residues located in the vestibules and pores of ion channels can play a major role in controlling the unitary conductance through an electrostatic mechanism (22-30). Such rings of fixed charge could increase the concentration of the permeating ions in the vestibules of the channels, leading to increased availability of ions to transit the selectivity filter, which would increase the unitary (single-channel) conductance.In this study we show by comparison of the sequence of BK channels to lower-conductance K ϩ channels and to a bacterial K ϩ channel (MthK) crystallized in the open state (31) that BK channels have a ring of eight negatively charged glutamate residues at the entrance to the intracellular vestibule that ...

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming α subunits and four modulatory β subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK α, and of the voltage-sensing domain TM helices S1–S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two β1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of α with two substituted Cys’s, one in S0 and one in S2, and β1 also with two substituted Cys’s, one in TM1 and one in TM2, resulted in two αs cross-linked by one β. Thus, each β lies between and can interact with the voltage-sensing domains of two adjacent α subunits.