Inward-rectifier K+ channels allow K+ to flow more efficiently into than out of the cell. This unusual property -- inward rectification -- enables these channels to maintain and regulate the resting membrane potential. By doing so, these channels accomplish many important biological tasks, e.g., controlling heart rate, modulating synaptic transmission and coupling insulin secretion to blood glucose levels. Inward rectification results primarily from a voltage-dependent channel blockade by intracellular cations such as Mg2+ and polyamines. In the ROMK1 inward-rectifier K+ channel the side-chain of an amino acid residue (171) within the second transmembrane region forms the Mg2+- binding site. In this proposed study, a combined molecular biological and electrophysiological approach will be used to establish the molecular architecture and physical location (depth) of the Mg2+-binding site. Quaternary ammoniums (QAs) block the ROMK1 channel by binding to the Mg2+-binding region and can thus be used to probe the dimensions of the region. A series of symmetric QAs of different sizes will be used to gauge the transverse dimension of the Mg2+-binding site. Second, a series of Bis-QAs, consisting of two ammonium groups connected by alkyl chains of various lengths, will be used to determine the depth of the Mg2+-binding site in the pore. The outcome of these studies will advance our understanding of the general molecular mechanisms of blocking-ion-induced rectification in inward-rectifier K+ channels.
