A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Frequency tuning of mechanical responses in the mammalian cochlea.

The search for mechanisms responsible for the high sensitivity and sharp frequency tuning of first-order auditory neurons has produced surprising results. The cochlea, the mammalian auditory receptor, responds to acoustic stimuli with a sharply frequency tuned, nonlinear vibration that enhances low level stimuli, but generates appreciable distortion. This highly sensitive mechanical response is achieved by an electro-mechanical feedback process in which outer hair cells reinforce cochlear motion at low stimulus intensities.

Frequency tuning of mechanical responses in the mammalian cochlea

The search for mechanisms responsible for the high sensitivity and sharp frequency tuning of first-order auditory neurons has produced surprising results. The cochlea, the mammalian auditory receptor, responds to acoustic stimuli with a sharply frequency tuned, nonlinear vibration that enhances low level stimuli, but generates appreciable distortion. This highly sensitive mechanical response is achieved by an electro-mechanical feedback process in which outer hair cells reinforce cochlear motion at low stimulus intensities

Concentraciones falsamente elevadas de hemoglobina glicosilada en 2 pacientes diabéticos con hemoglobina fetal persistente / Falsely high levels of glycosilated hemoglobin in 2 diabetic patients with persistent fetal hemoglobin

Two diabetic patients with unusual high levels of glycosilated hemoglobin measured by ion exchange chromatography are described. Further studies revealed a persistence of fetal hemoglobin in both cases. This condition produces falsely high levels of glycosilated hemoglobin, when ion exchange chromatography is used. These cases may be overtreated with risk of hypoglycemia. Patients with inappropiate levels of glycosilated hemoglobin should be investigated for hemoglobinopathies

Ion channels from the Bacillus subtilis plasma membrane incorporated into planar lipid bilayers.

Fusion of Bacillus subtilis plasma membrane vesicles with planar lipid bilayers induced the appearance of discrete current fluctuations characteristic of ion channels. These channels showed a wide range of conductances and kinetic behaviors. In 300 mM KCl, their conductances ranged from a few hundreds of pS to more than 1 nS, and most of them exhibited several sub-states. The channels poorly discriminated between small univalent anions and cations. Some of them showed voltage dependence and most of them presented a complex gating kinetics. The results are consistent with the hypothesis of the presence in the B. subtilis plasma membrane of pores composed of subunits that function cooperatively.

Activation of inositol trisphosphate-sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site.

Streaming potential measurements in Ca2+-activated K+ channels from skeletal and smooth muscle. Coupling of ion and water fluxes.

Streaming potentials arising across large-conductance Ca2+-activated K+ channels incorporated into planar lipid bilayers were measured. Ca2+-activated channels obtained either from skeletal muscle or from smooth muscle membranes were used. Streaming potentials were extracted from the current-voltage relationship for the open channel obtained in the presence of an osmotic gradient. The osmotic gradient was established by adding glucose to one side of the membrane. At 300 mM KCl, the average streaming potential was 0.72 mV/osmol per kg for t-tubule channels and 0.83 mV/osmol per kg for smooth muscle channels. Streaming potential values depend on KCl concentration, they decrease as KCl concentration increases, and the value obtained by extrapolation to zero KCl concentration is 0.85 mV/osmol per kg. Assuming that water and ions cannot pass each other, at least in a region of the channel, the streaming potential values obtained indicate that this region contains a minimum of two and a maximum of four water molecules. It is concluded that the channel has a narrow region with a length of 0.6-1.2 nm.