No abstract

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic ␤ cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.

SUMMARYThe effects of lidocaine have been investigated on electrical and contractile activity in guineapig ventricular cells in the absence and in the presence of ouabain. At low (therapeutic) doses, lidocaine induced a small reduction in action potential duration and contraction but had no effect on transient depolarizations or, under voltage-clamp conditions, on the transient inward currents. At much higher concentrations of lidocaine (> 500 /uM), where the fast inward sodium current was substantially blocked, there was also a marked reduction in the amplitude of the calcium current and accompanying phasic contraction. Again, lidocaine did not inhibit the transient depolarizations or transient inward currents. This suggests that there is no direct effect of lidocaine on the calcium-induced release of calcium from the sarcoplasmic reticulum and that lidocaine does not indirectly inhibit arrhythmic activity by reducing intracellular sodium in the isolated ventricular cell. Possible mechanisms for the antiarrhythmic action of lidocaine in whole heart are discussed.

The properties of the Ca2(+)-activated K+ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca2(+)-activated K+ channel which has the following characteristics: (1) single-channel conductance of 40-85 pS for outward currents in symmetrical K+ (150 mM) solutions. (2) inward currents of smaller conductance (10-50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K+ solutions, (3) channel activity dependent on the internal concentration of free Ca2+ and the membrane potential, (4) modification of the channel activity by internal adenosine 5' diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.