Two identical noninteracting sites in an ion channel revealed by proton transfer.

The functional consequences of single proton transfers occurring in the pore of a cyclic nucleotide-gated channel were observed with patch recording techniques. These results led to three conclusions about the chemical nature of ion binding sites in the conduction pathway: The channel contains two identical titratable sites, even though there are more than two (probably four) identical subunits; the sites are formed by glutamate residues that have a pKa (where K(a) is the acid constant) of 7.6; and protonation of one site does not perturb the pKa of the other. These properties point to an unusual arrangement of carboxyl side-chain residues in the pore of a cation channel.

Protein design of an HIV-1 entry inhibitor.

Human immunodeficiency virus type-1 (HIV-1) membrane fusion is promoted by the formation of a trimer-of-hairpins structure that brings the amino- and carboxyl-terminal regions of the gp41 envelope glycoprotein ectodomain into close proximity. Peptides derived from the carboxyl-terminal region (called C-peptides) potently inhibit HIV-1 entry by binding to the gp41 amino-terminal region. To test the converse of this inhibitory strategy, we designed a small protein, denoted 5-Helix, that binds the C-peptide region of gp41. The 5-Helix protein displays potent (nanomolar) inhibitory activity against diverse HIV-1 variants and may serve as the basis for a new class of antiviral agents. The inhibitory activity of 5-Helix also suggests a strategy for generating an HIV-1 neutralizing antibody response that targets the carboxyl-terminal region of the gp41 ectodomain.

Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel.

Divalent cation blockade of cGMP-gated channels in photoreceptor cells ensures the low open channel noise required for a highly sensitive visual transduction process. This study identifies a divalent cation-binding site in the pore of a retinal cGMP-gated channel expressed in Xenopus oocytes. Substitution of a specific glutamate residue by a neutral amino acid renders the channel insensitive to external Mg2+ and Ca2+ and affects the conduction of Na+. The mutated channels remain sensitive to internal divalent cations. These results place the glutamate residue in the ion conduction pathway close to the extracellular surface.

Communication barriers between older women and physicians.

Communication barriers between health care providers and older women are multifaceted and complex. The acute care, hospital-based orientation of the health care system tends to bypass the characteristic problems of older women who need services. Breakdown in communication originates both with the provider and the recipient. Some inherent changes of aging due to sensory loss may be a major factor. Decline in vision, hearing, and touch make communication difficult for both parties. Other deficits may occur due to disease processes such as diabetes and hypertension. A frequently ignored problem is that of the language barrier between laywomen and physicians. Use of jargon and a nonempathic interviewing style by the doctor tend to discourage free exchange of information. A third consideration is the many psychosocial factors which affect the behavior of older women and their relationship with providers. One element is the belief in negative stereotypes of women in general, and older women in particular. Fear of being labeled a "hypochondriac," a "nuisance," or a "crabby old woman" inhibits accurate reporting by patients. Attitudes toward doctors, especially male doctors, make some older women timid and fearful. Physician and patient alike may accept signs and symptoms of disease as a normal part of aging and may cause medically treatable problems to be overlooked. Finally, patient and physician priorities may differ widely. The belief by either party that wellness, prevention, and health promotion are not realistic goals for the older women may push the individual patient into premature frailty and disability which could otherwise be postponed.

Inhibition of the amorphous calcium phosphate phase transformation reaction by polyphosphates and metal ions.

The induction time for amorphous calcium phosphate (ACP) phase transformation was monitored at pH 7.4 and T = 25 degrees C with [Ca2+]o = [PO4]o = 4.0 X 10(-3) M, as a function of added crystal growth inhibitors Mg2+, Sr2+, Zn2+, pyrophosphate (PP), and tripolyphosphate (TPP). Metal ions increase the induction time for the initiation of the phase change reaction in the order Zn2+ less than Sr2+ less than Mg2+. For polyphosphates it was observed that both PP and TPP are potent inhibitors with TPP more effective than PP as expected. The combination of Mg2+ or Sr2+ and PP or TPP leads to a synergistic delay in the onset of the phase conversion. The greatest inhibition was observed for Mg2+ and TPP. Reaction solutions containing 2.0 X 10(-4) M Mg2+ and 4.0 X 10(-5) M TPP resulted in a 90% increase in the induction time over what would be anticipated from an additive effect from these species.

Influence of protein surface charge on the bimolecular kinetics of a potassium channel peptide inhibitor.

This study investigates the influence of a through-solution electrostatic interaction on the kinetics of ion channel blockade by the high-affinity peptide inhibitor Lq2. Membrane patches containing many Shaker K+ channels were removed from Xenopus oocytes and placed in a rapid perfusion chamber. Lq2 association and dissociation rate constants were determined from the relaxations to equilibrium blockade following rapid changes in toxin concentration. The association and dissociation rate constants were 8.5 x 10(7) M-1 s-1 and 0.71 M-1 s-1, respectively, in 100 mM NaCl solution, pH 7.1, at room temperature (21-23 degrees C). Charge-altering mutations introduced at position 422 on the ion channel affect toxin affinity in a manner consistent with a through-solution electrostatic interaction. The full effect of the charge mutations is expressed kinetically on the association rate; toxin dissociation remains unaltered. An electrostatic influence on the association rate alone is expected if diffusion of toxin up to (and away from) its receptor on the channel is fast compared to the rate of formation of short-range contacts that are necessary to produce the bound state.