The Oms66 (p66) protein is a Borrelia burgdorferi porin.

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

Pore formation by the cytotoxic islet amyloid peptide amylin.

Amylin is a 37-amino acid cytotoxic constituent of amyloid deposits found in the islets of Langerhans of patients with type II diabetes. Extracellular accumulation of this peptide results in damage to insulin-producing beta cell membranes and cell death. We report here that at cytotoxic concentrations, amylin forms voltage-dependent, relatively nonselective, ion-permeable channels in planar phospholipid bilayer membranes. Channel formation is dependent upon lipid membrane composition, ionic strength, and membrane potential. At 1-10 microM, cytotoxic human amylin dramatically increases the conductance of lipid bilayer membranes, while non-cytotoxic rat amylin does not. We suggest that channel formation may be the mechanism of cytotoxicity of human amylin.

Redistribution of the electric field within the pore contributes to the voltage-dependence of mitochondrial porin channel.

The effects of pH on the integral conductance and on the properties of single channels induced by porin from rat liver mitochondria in a lipid bilayer have been studied. When the membrane potential increases, the conductance of the multi-channel membrane decreases more sharply at acidic pH than at neutral or basic pH. The channel is shown to have several states with different conductance and selectivity. The number of levels and their conductance do not depend on pH, while the selectivity as well as the dependence of steady-state probabilities of different levels on the membrane potential are substantially affected by a pH change. This dependence curve steepens in the pH region where charges of carboxyl groups of aspartic and glutamic amino acids are neutralized. It is concluded that at neutral pH the channel gate is controlled by a great number of the positively and negatively charged groups. The high steepness of the conductance-voltage curve in the acidic region suggests that at least 60 positive charges participate in controlling the channel gate. This number, compared with that of the positively charged side chain amino acids per channel, according to the amino acid analysis of the porin, led us to conclude that almost all amino groups of the channel former must pass through the entire membrane potential difference upon random motion of the channel among the states. The assumption that channel closing leads to redistribution of the electric field within the pore, changing the energy of the charges on the voltage sensor, may be the only explanation of this phenomenon.

The gate of mitochondrial porin channel is controlled by a number of negative and positive charges.

Negatively charged carboxyl groups of mitochondrial porin have been converted into positively charged ones by means of reaction with water-soluble carbodiimide in the presence of ethylenediamine. Properties of channels formed in a planar lipid bilayer by native and modified porins are compared. Amidation has only little influence on the porin channel-forming activity as well as on the open-state conductance of the channel. However, the modification results in a significant enhancement of the voltage dependence of the channel gating and in an increase of the anionic selectivity. It is suggested that the voltage sensor of the porin channel gate is composed of a number of negative (greater than 14) and positive (greater than 22) charges.

Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors.

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.

[Stimulation of mitoplast respiration by mitochondrial porin]. / Stimuliatsiia dykhaniia mitoplastov mitokhondrial'nym porinom.

Mitochondrial porin (2 ng/ml) being added to the rat liver mitoplasts considerably stimulates the respiration in the third and uncoupled states. As the same effect was observed previously with the addition of outer membrane fraction to the mitoplast suspension, it is concluded that mitochondrial porin participates in regulation of the mitochondria respiration and, probably, is the natural activator of the ADP/ATP carrier function.

[Channel-forming membrane protein (32 kD) from brown fat mitochondria]. / Kanaloobrazuiushchii membrannyi belok (32 kD) iz mitokhondrii burogo zhira.

Isolated from brown adipose tissue mitochondria of newborn guinea pigs thermogenin forms ion channels in BLM which are analogous with mitochondrial porin channels.

[Ion channels formed in bilayer lipid membranes by large subunits of Na,K-ATPase and activated by ATP and p-nitrophenylphosphate]. / Ionnye kanaly, obrazuemye v bisloinykh lipidnykh membranakh bol'shimi sub''edinitsami Na,K-ATFazy i aktiviruemye ATF i p-nitrofenilfosfatom.

BLM modified by a large subunit of Na,K-ATPase is capable of forming ATP-dependent channels of conductivity in the presence of Na+ and K+ ions from the reaction medium eliminated the ATP effect, however, in this case the pNPP activated K+-conductivity is observed.

[Changes in K+ transport in the liver mitochondria of gophers in hibernation]. / Izmenenie transporta K+ v mitokhondriiakh pecheni suslikov pri zimnei spiachke.

Liver mitochondria of awake and hibernating squirrels maintain the same amount of K+ due to a decrease of K+-permeability during hibernation. The rates of DNP-stimulated K+-efflux and respiration-dependent K+-influx in mitochondria are diminished during hibernation. A two-fold increasing of K+-content and activation of K+-transport are observed at the arousing. Variations of K+-transport in mitochondria correspond to the changes in the channel-formed activity of K+-transporting protein isolated from the liver of this animals. This activity is maximal during arousal and is least during hibernation.