The ubiquity of cholesterol in cell membranes and changes in its concentration during development, aging and in various diseases suggest that it plays an important role in modulating cell function. We examined this possibility by monitoring the effects of cholesterol on the activity of the calcium-activated potassium (BK) channel reconstituted into lipid bilayers from rat brain homogenates. Increasing the cholesterol concentration to 11% of total lipid weight resulted in a 70% reduction in channel mean open time and a reduction of the open probability of the channel by 80%. Channel conductance was reduced by 7%. Cholesterol is known to change the order state and the modulus of compressibility of bilayers. These physico-chemical changes may be translated into an overall increase in the structural stress in the bilayer, and this force may be transmitted to proteins residing therein. By examining the characteristics of the BK channel as a function of temperature, in the presence and absence of cholesterol, we were able to estimate the activation energy based on Arrhenius plots of channel kinetics. Cholesterol reduced the activation energy of the BK channel by 50% for the open to closed transition. This result is consistent with an increased stress energy in the bilayer and favors the channel moving into the closed state. Taken together, these data are consistent with a model in which cholesterol induces structural stress which enhances the transition from the open to the closed state of the channel. We suggest that this is an important mechanism for regulating the activity of membrane-integral proteins and therefore membrane function, and that the concept of structural stress may be relevant to understanding the modulation of ion channel activity in cell membranes.

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Potassium channels govern duration and frequency of excitable membrane events and may regulate signals that are important in neuronal development. This study assesses the developmental expression of the large conductance Ca2+-dependent K+ channel in vivo and in vitro in rat cerebellum. In vivo, transcript levels for the Ca2+-dependent K+ channel (KCa) were shown by Northern analysis to increase during development, whereas transcript levels for the voltage-gated K+ channel Kv3.1, a delayed rectifier (KD), remained relatively constant. A comparable pattern was demonstrated by expression in Xenopus oocytes of poly(A)-enriched RNA isolated from postnatal rat cerebella. In cerebellar cultures, increased external K+ provided a simple manipulation of cell excitability that influenced KCa transcript levels during development. With low external K+ (5.3 mM), the levels of KCa channel transcript (assessed by semiquantitative PCR) remained constant throughout development. However, in culture medium that supported significant dendritic outgrowth (10 mM extracellular K+), an upregulation of KCa transcript level was observed similar to that seen in vivo. Tetraethylammonium (TEA; 1 mM) similarly enhanced KCa expression, suggesting that depolarizing stimuli increased KCa expression. The stimulatory effects of increased K+ or TEA on KCa expression required extracellular Ca2+ and were abolished in low external calcium (0.1 mM, buffered with EGTA), although morphological development and survival were not impaired. The regulation of KCa channel expression by depolarization and Ca2+ entry provides evidence of a logical feedback mechanism governing Ca2+ signals that may be significant in cerebellar development.

We have recently shown (Chang et al., 1995) that lipid-channel interactions, exemplified by the effects of cholesterol on the calcium-activated potassium (BK) channel, profoundly affect channel properties. The present study further explores such interactions by monitoring changes in BK channel behavior after reconstitution into bilayers where the size of phospholipid (PL) headgroups is increased and where the freedom of motion (inverse order) of fatty acid chains is incremented. Increasing the PL headgroup cross-sectional area, from that of N-meth-DOPE to that of DOPC (an increase from ca. 60 to 70 A2), is associated with a doubling of the channel mean opentime. Channel conductance, however, was unaffected. Increasing the order of the fatty acid chains, from that of DOPE to POPE and to that of DEPE, had no significant effect on channel properties (at 22 degrees C). We interpret the changes reported here to reflect lipid-protein interactions through the induction of structural stress related to the headgroup structures of phospholipids.

Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis. Rapid and simple assays for the identification of individual Leptospira species are currently not available. For identification of individual Leptospira species, PCR primers that detect the ompL1 gene sequence for the majority of pathogenic leptospires were developed in this study. The primer pairs detect Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira santarosai, Leptospira weilii and Leptospira noguchii, without cross-reacting with other Leptospira species. The development of the primers revealed a divergence of the ompL1 gene within L. interrogans, splitting this species into two separate groups. The species-specific primers will be especially useful in epidemiological studies and disease outbreak investigations for the detection of Leptospira species in human, animal and environmental samples.