Binding of Ca(2+) to Glutamic Acid-Rich Polypeptides from the Rod Outer Segment

Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca(2+) from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca(2+) to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca(2+) binding was investigated by means of a Ca(2+)-sensitive electrode. In all cases, Ca(2+) binds with low affinity; the half-maximum binding constant K(1/2) ranges from 6 to 16 mM. The binding stoichiometry between Ca(2+) ions and carboxylic groups is ∼1:1; an exception is GARP2, where a binding stoichiometry of ∼1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca(2+) binding

A family of hyperpolarization-activated channels selective for.

Proton (H + ) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few pro- ton channels have been identified so far. Here, we identify a fam- ily of voltage -gated ?pacemaker ? channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpo- larization and conducts protons into the cytosol. Surprisingly, pro- tons permeate through the channel ?s voltage -sensing domain, whereas the pore domain is nonfunctional. Key to proton perme- ation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional do- mains, HCNL1 evolutionarily adapted to a low-Na + freshwater en- vironment to conserve sperm ?s ability to depolarize