1. Short segments of interlobular duct were microdissected from guinea-pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]1) were measured by microfluorometry in duct cells loaded with either the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or the sodium-binding benzofuran isophthalate (SBFI).2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4C1 pulse technique to acid load the cells. In the absence of HCO3-/CO2, the recovery of pHi was Na+ dependent, abolished by 0-2 mm amiloride and by 10 am N-methyl-N-isobutylamiloride and was therefore attributed to Na+-H+ exchange.3. In the presence of HC03-/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride-insensitive component was abolished by 0 5 mm H2DIDS and unaffected by depletion of intracellular Cl-and was therefore attributed to Na+-HCO03 cotransport.4. Stimulation with 10 nm secretin did not cause a significant change in pHi despite a significant increase in HC03-efflux. However, in the presence of secretin, addition of 0 5 mm H2DIDS caused a decline in pHi that was three times more rapid than that obtained with 0-2 mm amiloride.5. In secretin-stimulated ducts, Na+ uptake increased when HC03-/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mm H2DIDS. 6. We conclude that Na+-HCO -cotransport contributes approximately 75% of the HC03-taken up by guinea-pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HC03-efflux at the luminal membrane and electrogenic

A model is developed for ionic conduction in the sheep cardiac sarcoplasmic reticulum ryanodine receptor channel based on Eyring rate theory. A simple scheme is proposed founded on sin#e-ion occupancy and an energy profile with four barriers and three binding sites. The model is able to quantitatively predict a large number of conduction properties of the purified and native receptor with monovalent and divalent cations as permeant species. It suggests that discrimination between divalent and monovalent cations is due to a high affinity central binding site and a process that favors the passage of divalent cations between binding sites. Furthermore, differences in conductance among the group Ia cations and among the alkaline earths are largely explained by differing affinity at this putative central binding site.

SUMMARY1. The ryanodine receptor protein of sheep cardiac muscle sarcoplasmic reticulum membranes functions as a ligand-regulated ion channel following solubilization with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate); purification by density gradient centrifugation, reconstitution into proteo-liposomes and incorporation into planar phospholipid bilayers.2. In the absence of divalent cations, measurable conductance is observed with the group la cations and with some larger organic cations. In symmetric 210 mM solutions the following conductance sequence was determined: K+ > Rb+ NH4+ > Na+ = Cs+ > Li+ > Tris+.3. Other organic cations, e.g. TEA+, do not produce measurable current under these conditions. 4. Single-channel conductance saturates with increasing ionic activities of K+, Na+ and Li+. Saturation curves are described by Michaelis-Menten kinetic schemes with the following values of maximal conductance and apparent dissociation constant: K+ 900 pS, 19-9 mM; Na+ 516 pS, 17-8 mM; Li' 248 pS, 9-1 mM.5. The channel displays only minor differences in permeability amongst the group la cations. Relative permeability, monitored under bi-ionic conditions, yields the following sequence: Na+, 1-15 > K+, 1-00 = Li+, 0 99 > Rb+, 0-87 > Cs+, 0-61. Under similar conditions the permeability ratio of NH4+ to K+ was found to be 1-32 and that for Tris+ to K+ was 0-22.6. The K+ conductance is reduced by low concentrations of the impermeant cation TEA+. Block appears as a smooth reduction in single-channel current amplitude and the degree of block is dependent upon applied voltage. These observations are consistent with a single-site blocking scheme in which TEA+ has access to a site within the voltage drop of the channel from only the cytosolic face of the channel protein and interacts with a site located approximately 90% of the electrical distance across the channel. The zero-voltage dissociation constant for TEA+ block is 50 mM.7. Single-channel conductance measurements in mixtures of K+-Na+ and K+-Li+ reveal no anomalous behaviour as the mole fraction of the ions is varied.8

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca2+-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group la monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group la monovalent cations (K +, Na +, Cs § Li +) have values of fractional conductance in a narrow range (0.60-0.66). With divalent cations fractional conductance is considerably lower (Ba 2+, 0.22 and Sr 2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum CaZ+-release channel produces a conformational Address correspondence to Dr. Alan Williams, Department of Cardiac Medicine, National Heart and Lung Institute, University of London, Dovehouse Street, London SW3 6LY, UK. 426THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 104 9 1994 alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group la monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation. Small cations such as ammonia respond to ryanodine-induced alterations of the conduction pathway in much th...

Variations in permeant ion concentration such as may occur in physiological and pathophysiological states may significantly affect the quantity of Ca2+ released from the cardiac sarcoplasmic reticulum.

The purified Ca(2+)-release/ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) functions as a calcium-activated cation-selective channel under voltage clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effect of large tetraalkyl ammonium (TAA) cations, (CnH2n+1)4N+ (n = 4 and 5) on monovalent cation conduction. These cations modify the conductance of the receptor channel at positive holding potentials from the cytosolic side of the channel. Under these conditions, openings are resolved as a mixture of normal full amplitude events and events of reduced conductance. The amplitude of the reduced conductance state is a fixed proportion of the normal open state. As a proportion of all open events, the occurrence of the tetrabutyl ammonium (TBA+) related subconductance state increases with concentration and increasingly positive holding potential. The TBA+ related subconductance state displays similar conduction properties to the unmodified channel; with a linear current-voltage relationship, a similar affinity for K+ and voltage-dependent block by TEA+. A method was used to quantify the voltage dependence of the occurrence of the TBA+ effect, which yielded an effective gating charge of 1.66. A second method based on kinetic analysis of the voltage dependence of transitions between the full open state and the TBA+ related subconductance state produced a similar value. In addition, this analysis revealed that the bulk of the voltage-dependence resided in the off rate. TBA+ related subconductance events, expressed as a proportion of all open events, saturated with increasing TBA+ concentration. Kinetic analysis revealed that this could be entirely accounted for by changes in the on rate. Tetrapentyl ammonium (TPeA+) causes a qualitatively similar effect with a subconductance state of lower amplitude. The voltage-dependence of the effect was comparable to that displayed by TBA+. These findings are interpreted as a form of partial block in which more than one large TAA cation binds at the extremity of the voltage drop to produce an electrostatic barrier for ion translocation.

The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (CnH2n+1)4N+ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1-3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA+) and 90% for tetraethyl ammonium (TEA+) and tetrapropyl ammonium (TPrA+) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA+ increases the voltage dependence of block to a level similar to that of TEA+ and TPrA+. The zero-voltage dissociation constant (Kb(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a Km of 9.8 +/- 1.7 mM (mean +/- SD) at 40 mV. Larger TAA cations (n = 4-5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.

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