Article Title: The Ca2+-activated K+ current of human sperm is mediated by Slo3
Figure Lengend Snippet: Lack of homology among Slo3 sequences in the ligand-sensing cytosolic domain. ( A ) Alignments of human Slo1 and Slo3 from various species are shown for the membrane-associated, pore-forming part of the channels, indicating the relatively high extent of conservation through this part of the Slo3 protein. The Slo1 N-terminus is omitted to minimize effects of S0–S1 linker gaps on the alignment. Slo1 numbering starts from amino acids MDAL. Tick marks below each segment of residues counts every 10 residues in human Slo3. ( B ) Alignments of human Slo1 and Slo3 from various species are shown for the cytosolic gating ring domain beginning with the conserved sequence at the beginning of the first RCK domain. Blue highlights residue differences between human Slo1 and human Slo3. Yellow highlights differences of Slo3 of various species to human Slo3. Alignments were generated by Clustal 1.2.0 and minor adjustments were made based on structural considerations ( Leonetti et al., 2012 ). Above the residues, the correspondence of particular amino acid segments to structurally defined α-helical and β-strand segments is shown based on Leonetti et al. (2012) . In red, residues or segments identified in Slo1 or Slo3 isoforms which are implicated in ligand-sensing or species-specific functional differences are highlighted. Although extensive information is available regarding loci important in ligand-sensing in Slo1, such information for Slo3 remains lacking. Numbers identify the following: 1, the sequence of residues termed the Ca 2+ bowl ( Schreiber and Salkoff, 1997 ), for which there is good correspondence of mutations affecting Ca2+-dependent function ( Bao et al., 2004 ) and coordination of density in a crystal structure ( Yuan et al., 2012 ); 2, the D367 residue implicated in the role of the RCK1 domain in Ca 2+ -dependent activation ( Xia et al., 2002 ) which is clearly distinct from Ca 2+ -bowl dependent activation ( Zeng et al., 2005 ); 3, the M513 residue, which also affects Ca 2+− dependent activation involving the RCK1 domain ( Bao et al., 2002 ), but probably is not involved in ligand coordination; 4, residues E374 and E399 which have been implicated in low affinity effects of divalent cations, specifically Mg 2+ ( Shi et al., 2002 ; Xia et al., 2002 ; Yang et al., 2006 ); 5, residue E535 which may also be involved in Ca 2+ coordination in RCK1 ( Zhang et al., 2010 ); 6, residues H365 and H394, which have been implicated in proton-dependent activation of Slo1 and also influence Ca 2+ -dependent activation when protonated ( Hou et al., 2008 ); 7, H417 and segment 368–475, which influence pH-sensing in mouse Slo3 (Zhang et al., 2006); 8, segment 495–515 in bovine Slo3 which accounts for part of the different in functional properties between mouse Slo3 and bovine Slo3 ( Santi et al., 2009 ). Illustrated sequences and accession numbers include: HsSlo1 ( Homo sapiens ), NP_001154824, Gene ID 3778; HsSlo3 ( Homo sapiens ), NP_001027006, Gene ID 157855; MmSlo3 ( Mus musculus ), NP_032458, Gene ID 16532; RnSlo3 ( Rattus norvegicus ), XP_006253398, Gene ID 680912; TcSlo3 (Tupaia chinensis, Chinese tree shrew), XP_006171561, Gene ID 102493286; CcSlo3 ( Condylura cristata , star-nosed mole), XP_004682520, Gene ID 101620543; CfSlo3 (Canis lupus familiaris), XP_539971, Gene ID 482856; BtSlo3 (Bos taurus), NP_001156721, Gene ID 524144; OaSlo3 ( Ovis aries , sheep), XP_004021821, Gene ID 10110209. DOI: http://dx.doi.org/10.7554/eLife.01438.019
Article Snippet: DMNP-EDTA and Ca2+ indicators were purchased from Invitrogen (Carlsbad, CA, USA).
Techniques: Sequencing, Generated, Functional Assay, Activation Assay