MthK is a Ca2+-gated K+ channel whose activity is inhibited by cytoplasmic H+. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H+ inhibition and Ca2+-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca2+] (ranging from nominally 0 to 30 mM) at a given [H+], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca2+] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH changed significantly as a function of [H+] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca2+ was not H+ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H+] raised the EC50 for Ca2+ activation by ∼4.7-fold per tenfold increase in [H+], displaying a linear relation between log(EC50) and log([H+]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H+ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca2+ binding and channel opening. We can account for the Ca2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca2+ allosterically activates MthK, whereas H+ opposes activation by destabilizing the binding of Ca2+.

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of "natively unfolded" proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.

Regulator of K þ conductance (RCK) domains control the activity of a variety of K þ transporters and channels, including the human large conductance Ca 2þ -activated K þ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca 2þ -gated K þ channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca 2þ . Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca 2þ -binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca 2þ -binding sites, resulting in a stoichiometry of 24 Ca 2þ ions per channel, is consistent with the steep relation between [Ca 2þ ] and MthK channel activity. Comparison of Ca 2þ -bound and unliganded RCK domains suggests a physical mechanism for Ca 2þ -dependent conformational changes that underlie gating in this class of channels.calcium | lipid bilayer | cooperativity R egulator of K þ conductance (RCK) domains are structurally conserved ligand-binding domains that control the activity of a diverse array of K þ channels and transporters (1-3). Many prokaryotic RCK domains contain a conserved sequence motif for binding of nucleotides (NAD þ or ATP) (4, 5). In some prokaryotic and most of the known eukaryotic RCK-containing K þ channels, however, the nucleotide binding motif is absent, and these channels are modulated by cytoplasmic ions such as Na þ , H þ , or Ca 2þ (6-12).MthK is a prototypical RCK-containing K þ channel that has provided insight toward the structural basis of ion channel gating by RCK domains (2,13,14). In MthK, binding of Ca 2þ to an octameric ring of RCK domains (the gating ring), which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K þ conduction (2, 15, 16). Based on X-ray structures of the Ca 2þ -bound MthK channel and the unliganded MthK gating ring (17), it has been hypothesized that the principal Ca 2þ -dependent conformational change is initiated by the movement of a Glu side chain (E212) at a single Ca 2þ -binding site within each RCK domain ( Fig. 1A; site 1, formed by D184, E210, and E212), followed by subsequent movement of a nearby Phe side chain (F232). However, the conformational changes in the immediate vicinity of site 1 are relatively subtle compared to apparent conformational changes in other regions of the RCK domains (17); thus the mechanism by which Ca 2þ binding at site 1 modulates channel gating is unclear.To gain insight toward mechanisms underlying Ca 2þ -dependent conformational changes in the MthK RCK domain, we probed MthK structure and function using electrophysiology and crystallography. Our results demonstrate that whereas site 1 contributes energetically to Ca 2þ -dependent gating, charge-neutralization of the key Ca 2þ -coordinating residues at this site do not eliminat...