Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels

Contributions of the C-terminal domain to gating properties of inward rectifier potassium channels

AbstractTwo inward rectifier potassium channels, the G protein-dependent GIRK1 and the G protein-independent BIR10, display large differences in rectification and macroscopic kinetics. A chimeric channel was constructed in which the putative intracellular carboxy-terminal domain of the G protein-dependent channel replaced the corresponding domain of the G protein-independent channel. The chimeric channel conducted potassium ions without the requirement of activated G proteins, yet displayed activation and deactivation kinetics and rectification properties similar to those of the G protein-dependent channel. The results demonstrate that structural elements in the C-terminus can independently control gating but not G protein signal transduction. The voltage dependence, time course, and kinetics of gating suggest a mechanism in which the pore may be occluded by reversible interactions with charged residues in the C-terminus

Solubilization and characterization of σ-receptors from guinea pig brain membranes

The σ-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for σ-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective ρ-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 ± 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(l-propyl)piperidine {(+)-[3H]3-PPP} (KD = 36 ± 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a ρ-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the σ-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macro-molecular complex