P-C2-12 Phosphatidylserine and purine nucleotides regulate colonic BKCa channel activity

C2 Gated ion movement and pumping PC2-09

P-C2-10 ON THE CONDUCTANCE OF CHANNELS UNDERLYING I, IN DRG NEURONS RAESA’, VAN DEN BERGm, VAN HARTEVELT f, VAN BOGAERTPP’. ’ Dept.of Biochem.-PhysioL-Gen., Univ.of Antwerp(BE), ’ Lab.of Physiology, Univ.of L&den(NL)

CONTRIBUTION OF Ix, TO THE CARDIAC ELECTRICAL RESPONSE TO METABOLIC INHIBITION RIOUX Y,.RUIZ PETRICH E. Dept. Phyaol.andBiophys., Univ.of Sherbrooke (CA)

Purpose: Methylisobutyl amiloride (MIA) lengthensthe action potential and protects against repetfbsioninduced arrhythmia in rat hearts. MIA also reduces the action potential shortening produced by hypoxia in rabbit hearts. We investigatedthe effectsof MIA on ionic currentsin rabbit cardiomyocytes. Methods: Myocyteswere isolated from rabbit left ventricle and septum by enzymatic dissociation. Ionic currentswere measuredwith the patch clamp techniquein the whole cell configuration. I,, was determinedasthe steadystatecurrent at the end of 400 ms pulses between -110 and +lO mV. IckL elicited by pulsesbetween -40 and +40 mV from a V, of -50 mV was also measured. Results: MIA produced a progressive depression of I,, that reached a steady state after 10 to I5 min. This effect was dose dependant. In the potential range where I,, contributes to action potential repolarization, 1 /*M MIA reduced the currentby27.4~1.2%andlO~Mby41.9~1.5 %. The Ca” current was not significantly modified. Conclusions: Our data show that: a) the action potential lengthening produced by MIA can be exnlained bv the reduction of I,, and b) the an&atrhyth&c effect of MIA is-not due’to a reducedCa%entrythroughvoltage gated channels.

Purpose: The single channel conductance underlying the hyperpolarization-activatedcurrent (IJ is too small to detectchannelevents.Therefore, we used stationary noise analysis to estimate its magnitude at room temperature. Methods: Noise measurements(0.2 - 800 Hz) simultaneouslywith current recordingswere performed on cultured mouse DRG neurons in the cellattached-patchconliguration(70 mM K+in the patch pipette). Various noise models were fitted to the frequency spectra and evaluated with information criteria. Results: The spectraldensity of I, fluctuations was best described by the sum of 2 Lorentzians, a l/f noise component and a constant level at high frequencies. From the noise variance produced by the Lorentzian noises the single channel conductancey was calculatedas 0.60 f 0.10 pS (A sem, n=17). Conclusions: Noise analysis yielded a single channelconductanceof sucha magnitude explaining the lack of channeleventsin our current recordings. Under physiological conditions (5 mM K*) the value of y will presumablybe as low as f 0.1 pS.

P-C2- 11 lNCORPOBATlON OF BACTERIORHOSOLID SUPPORTED DOPSIN IN MEMBRANES STElNEM C., JANSHOFF A., HGHN F., SlEBERM., GALLA H.-J. lnstitutfir Biochemie. Universitiit Munster.Germany Purpose: As a fundamental requirement in constructing biosensor devices based on solid supported membranes (SSMs) with incorporated proteins it is necessary to characterize the immobilized system. SSMs are well suited for electrochemicaland optical analysis and exhibit long-term stability as well as high reproducibility. In this study we present the incorporation of the well-known membrane protein bacteriorhodopsin (BR) as a light inducedproton pump. Methods: SSMs were attached to gold substrates with self-assemblytechniques and characterized by impedance spectroscopy. The light induced photocurrent of the membrane contlmed BR was measuredwith a current amplifier. Results: The different SSMs exhibit high stability and nearly complete electrode coverage. Incorporation of BR was possible by fusion of proteoliposomeson the preformed monolayer. BR maintainsnormal activity within the membrane. Conclusions: SSMs are well suited for the study of membraneproteins retaining native btion and show good prerequesites for biosensor development.

P-C2-12 PHOSPHA’-QDYLSjWINE AND PURINE NUCLEUTWS REGULATE COLONIC BI& CHAN@EL AC’l’ItlTY. WACHTER C, ZEUTXEN T, KLAERKE DA Dept.of MedicalPhysiology, The PanumInstitute,Univ. of Copenhagen (DK) Purpose: In this study we investigate the influence of phos@olipids on the activity of large conductance, Ca -activated K’ channels (Bl& channels) from the basolateral membrane of rabbit distal colon epithelium surfacecells. Methods: Bl$ channels are incorporated into planar lipid bilayers by fusion of basolateral plasma membrane vesicles, and channel activity is measuredusing a patch clamp amplifier. Results: Channel open probability, PO,decreases to a minimum shortly after incorporation into bilayers consisting of equal amounts of phosphatidylethanolamine and U’E) phosphatidylcholine (PC). Raising the free Ca” concentrations restores Po only transiently, whereas Po can be permanently restored by 0.1 mM puke nucleotides. In contrast, POis stable for hours in bilayers consisting of PE and phosphatidylserine (PS). 10% PS in PEfpC bilayersis sufficient to stabilize channel activity. Conclusions: We suggestthat Bl(c* channels are activated by the presenceof PS in the vicinity of the channel protein and by direct bindin of nucleotides. Channel “rundown” in Pl&PC bilayersmay tbcrefbre be the result of substitution of PS contaiasd in the ariejaial cell membrane with PC from the surrounding bilayer. 102