Mapping the BKCa Channel's “Ca2+ Bowl”

Abstract

There is controversy over whether Ca2+ binds to the BKCa channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca2+ sensitivity act at the point of Ca2+ coordination. One region in the intracellular domain that has been implicated in Ca2+ sensing is the “Ca2+ bowl”. This region contains many acidic residues, and large Ca2+-bowl mutations eliminate Ca2+ sensing through what appears to be one type of high-affinity Ca2+-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca2+ bowl that are most important for Ca2+ sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca2+ binding to a fusion protein that contains a portion of the BKCa channel's intracellular domain. Thus, much of our data supports the conclusion that Ca2+ binds to the BKCa channel's intracellular domain, and they define the Ca2+ bowl's essential Ca2+-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca2+ sensing and those that disrupt Ca2+ binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca2+ bowl may be in a nonnative conformation.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

“…However, the well characterized block effect of Ba 2ϩ at the BK channel pore (55)(56)(57)(58) (24). This finding was in agreement with previous electrophysiological investigations (29,34,35) as well as biochemical investigations of the calcium bowl region (30,32,60) and the whole RCK2 domain (33). Although it has yet to be confirmed in atomic structures, electrophysiological investigations support the existence of a second micromolar-affinity Ca 2ϩ -bindng site in the RCK1 domain (34 -38); indeed, purified RCK1 domains in solution assemble into homo-octameric gating ring-like structures that sense micromolar Ca 2ϩ in solution (47).…”

Hydrophobic Interface between Two Regulators of K+ Conductance Domains Critical for Calcium-dependent Activation of Large Conductance Ca2+-activated K+ Channels

“…The Ca 2ϩ bowl is composed of a series of Asp residues and binds Ca 2ϩ with micromolar affinity. Mutations here have been shown to cause positive shifts in the conductance-voltage (G-V) relationship at constant [Ca 2ϩ ], which are similar to those observed with the wild-type channel when [Ca 2ϩ ] is lowered (22,23,26,30,31). The RCK domain is found primarily in prokaryotic ligandgated K ϩ channels and in some bacterial K ϩ uptake and efflux systems, in which it is also called the K ϩ transport nucleotidebinding (KTN) domain (25,(32)(33)(34).…”

The RCK2 domain of the human BK Ca channel is a calcium sensor

“…A well studied Ca 2ϩ -binding site corresponds to a C-terminal region that includes five consecutive negatively charged aspartates (D894-D898), christened the ''Ca bowl'' by the Salkoff laboratory (16,17). The Ca bowl binds Ca 2ϩ with high affinity (18)(19)(20)(21) and strongly contributes to the channel's Ca 2ϩ sensitivity (18)(19)(20) [supporting information (SI) Fig. 6].…”