Inward current activated by hyperpolarization (ih) was dissected from the K-current by the difference in its activation voltage range and the selective blocking effect of Ba2+ on the K-current. The ih shows little specificity to any particular ion, and its reversal potential was -25 mV. The current system can be expressed well by Hodgkin-Huxley type kinetics. The time constant of ih ranged from 2-4 s at about -70 mV, but it became shorter at about -10 mV. The ih began to activate at -50 mV and fully saturated at about -100 mV. The fully activated current-voltage relation shows no rectifying property. Activation and deactivation time courses were fitted by a single exponential with the same time constant at a given membrane potential. Although ih plays only a small role during the normal action potential in the isolated preparation, it plays a significant role in keeping the pacemaker cell at a low membrane potential

The pacemaker activity of the S-A node cell was explained by reconstructing the pacemaker potential using a Hodgkin-Huxley type mathematical model which was based on the reported voltage clamp data.In this model four dynamic currents,the sodium current,ZNa,the slow inward current,is,the potassium current,iK,and the hyperpolarization-activated current,ih,in addition to a time-independent leak current, the current voltage relationship,and the voltage clamp experiment in normal Tyrode solution of the rabbit S-A node.Furthermore,the changes of activity induced by the potassium current blocker Ba2+, by applying constant current,acetylcholine,and epinephrine were also reconstructed. It was strongly suggested that the pacemaker depolarization in the S-A node cell is mainly due to a gradual increase of is during diastole,and that the contribution of ig is much less compared to the potassium current iK2 in the Purkinje fiber pacemaker depolarization.The rising phase of the action potential was due to is and the plateau phase is determined by both the inactivation of is and activation of ik.Automatic excitation is one of the characteristic features of the myocardium. This property is solely attributable to the pacemaker activity of the sino-atrial node(S-A node)cells in the intact heart,although other myocardia can also initiate a spontaneous action potential under various experimental,as well as pathological conditions. Spontaneously beating cells are characterized by having a slow diastolic depolarization (DRAPER and WEIDMANN,1951;TRAUTWEIN and ZINK,1952; WEST,1955).Ionic mechanisms underlying pacemaker depolarization have been extensively studied by voltage clamp experiments,and the result has always shown a gradual decay of K conductance on repolarization after a depolarizing clamp pulse.Pacemaker depolarization generated mainly by a spontaneous decrease of K conductance is confirmed in the mathematical model of cardiac excitation

Clp͞Hsp100ATPases comprise a large family of ATP-dependent chaperones, some of which are regulatory components of two-component proteases. Substrate specificity resides in the Clp protein and the current thinking is that Clp proteins recognize motifs located near one or the other end of the substrate. We tested whether or not ClpA and ClpX can recognize tags when they are located in the interior of the primary sequence of the substrate. A protein with an NH 2-terminal ClpA recognition tag, plasmid P1 RepA, was fused to the COOH terminus of green fluorescent protein (GFP). GFP is not recognized by ClpA or ClpX and is not degraded by ClpAP or ClpXP. We found that ClpA binds and unfolds the fusion protein and ClpAP degrades the protein. Both the GFP and RepA portions of the fusion protein are degraded. A protein with a COOH-terminal ClpX tag, MuA, was fused to the NH 2 terminus of GFP. ClpXP degrades MuA-GFP, however, the rate is 10-fold slower than that of GFP-MuA. The MuA portion but not the GFP portion of MuA-GFP is degraded. Thus, a substrate with an internal ClpA recognition motif can be unfolded by ClpA and degraded by ClpAP. Similarly, although less efficiently, ClpXP degrades a substrate with an internal ClpX recognition motif. We also found that ClpA recognizes the NH 2-terminal 15 aa RepA tag, when it is fused to the COOH terminus of GFP. Moreover, ClpA recognizes the RepA tag in either the authentic or inverse orientation. molecular chaperones ͉ proteases ͉ ClpA ͉ ClpX ͉ Hsp100 C lp͞Hsp100 ATPases comprise a large family of homologous ATPases with ATP-dependent molecular chaperone activity. They participate in many cellular functions, including DNA replication, tolerance to heat stress, control of gene expression, and protein degradation (1, 2). Several Clp proteins, including Escherichia coli ClpA, ClpB, and ClpX, and yeast Hsp104, act in protein remodeling reactions in vitro such as disassembly of complexes and the reactivation and disaggregation of denatured proteins (3-7). In addition, some Clp proteins play a direct role in protein degradation by associating with a proteolytic component to form ATPdependent proteases. For example, ClpA, ClpX, and HslU of E. coli are the ATPase components of ClpAP, ClpXP, and HslUV, respectively (8,9).Structural studies have shown that Clp ATPases self-assemble into oligomeric rings in the presence of ATP or nonhydrolyzable ATP analogs (10, 11). When associated with a proteolytic component, the ATPase rings are located at either or both ends of the proteolytic core forming a structure resembling the eukaryotic 26S proteasome (10, 12). The crystal structures of ClpP and HslV show that the proteolytic active sites are in an internal chamber formed by two stacked rings of identical subunits (13,14). Access to the proteolytic chamber appears to be through narrow pores at either end of the stacked rings. However, the pores, which measure about 10 Å in ClpP (13), are not wide enough to allow the passage of globular proteins.The pathway of proteolysis suggested by the struc...