Inhibition by nickel of the L-type Ca channel in guinea pig ventricular myocytes and effect of internal cAMP

The characteristics of nickel (Ni) block of L-type Ca current ( I Ca,L) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37°C in the absence and presence of 100 μM cAMP in the pipette solution. Ni block of peak I Ca,L had a dissociation constant ( K d) of 0.33 ± 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the K d was 0.53 ± 0.05 mM ( P = 0.006). Ni blocked Ca entry via Ca channels (measured as I Ca,L integral over 50 ms) with similar kinetics ( K d of 0.35 ± 0.03 mM in cAMP-free solution and 0.30 ± 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of I Ca,L was largely use independent. Ni (0.5 mM) induced a positive shift (4 to 6 mV) in the activation curve of I Ca,L. The block of I Ca,L by 0.5 mM Ni was independent of prepulse membrane potential (over the range of −120 to −40 mV). Ni (0.5 mM) also induced a significant shift in I Ca,Linactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 μM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block I Ca,L and Ca entry into cardiac cells.

Ion–Ion Interactions at the Selectivity Filter

In the Kv2.1 potassium channel, binding of K+ to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na+ current, K+ interacts with the TEA modulation site at concentrations ≤30 μM. In this paper, we further characterized the TEA modulation site and examined how varying K+ occupancy of the pore influenced the interaction of K+ with this site. In the presence of high internal and external [K+], TEA blocked 100% of current with an IC50 of 1.9 ± 0.2 mM. In the absence of a substitute permeating ion, such as Na+, reducing access of K+ to the pore resulted in a reduction of TEA efficacy, but produced little or no change in TEA potency (under conditions in which maximal block by TEA was just 32%, the IC50 for block was 2.0 ± 0.6 mM). The all-or-none nature of TEA block (channels were either completely sensitive or completely insensitive), indicated that one selectivity filter binding site must be occupied for TEA sensitivity, and that one selectivity filter binding site is not involved in modulating TEA sensitivity. At three different levels of K+ occupancy, achieved by manipulating access of internal K+ to the pore, elevation of external [K+] shifted channels from a TEA-insensitive to -sensitive state with an EC50 of ∼10 mM. Combined with previous results, these data demonstrate that the TEA modulation site has a high affinity for K+ when only one K+ is in the pore and a low affinity for K+ when the pore is already occupied by K+. These results indicate that ion–ion interactions occur at the selectivity filter. These results also suggest that the selectivity filter is the site of at least one low affinity modulatory effect of external K+, and that the selectivity filter K+ binding sites are not functionally interchangeable.

Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 ± 2.1 pS ( n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 μM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.

Plasmin Inhibition of Thrombin-induced Platelet Aggregation

SummaryThe effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10–20 μM, plasmin (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin’s inhibition of aggregation.Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction.Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin -treated platelets, upon centrifugation and treatment with SBTI.With the use of a “cold initiation” technique, the release by thrombin of 46.7 ± 6-7 (mean ± SEM) μg of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by SBTI) aggregated normally upon addition of as little as 10 μg human plasma fibrinogen per mg platelet protein.It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.