It has been known for more than three decades that outward Kir currents (I K1 ) increase with increasing extracellular K ؉ con- Inward rectifier K ϩ channels (Kir) 3 channels are important in maintaining stable resting membrane potentials, controlling excitability, and shaping the initial depolarization, as well as the final repolarization of action potentials in many cell types, including heart cells (1-6). The physiological functions of Kir channels are closely related to their unique inward rectification mechanism, which allows inward currents to pass through the channel more easily than outward currents (6). Although small, the outward I K1 plays a crucial role in controlling membrane excitability and action potential duration. The gain or loss of outward I K1 in the heart can lead to re-entry or arrhythmia, respectively (7). In contrast to normal conditions, in which the extracellular is increased, the outward I K1 increases, despite a reduction in the electrochemical gradient (1, 11). Accordingly, increases in I K1 result in reduced excitability, slow conductance, and abbreviation of the refractory period, and thereby predispose the heart to re-entrant ventricular arrhythmias, the leading cause of death from coronary artery disease (12). The [K ϩ ] o dependence of the outward I K1 thus might be important for regulating the physiological and pathological functions of the heart. Despite the importance of this long known [K ϩ ] o dependence of the outward I K1 , the underlying mechanism has remained unclear.Previous studies have shown that the dependence of Kir channel activity on membrane potential (V m ) shifts in parallel with V m -E K (1-6), where E K is the equilibrium potential for K ϩ . Thispropertyhasbeenpreviouslyattributedtothedrivingforcedependent block of Kir2.1 channels by intracellular blockers (13,14). According to this hypothesis, known as the blockingparticle model, cytoplasmic blockers are dragged into or pushed out of the channel pore by the outward or inward flux of K ϩ , respectively.

Outward currents through Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells, and such currents are subjected to voltage-dependent block by intracellular Mg2+ and polyamines that bind to both high- and low-affinity sites on the channels. Under physiological conditions, high-affinity block is saturated and yet outward Kir2.1 currents can still occur, implying that high-affinity polyamine block cannot completely eliminate outward Kir2.1 currents. However, the underlying molecular mechanism remains unknown. Here, we show that high-affinity spermidine block, rather than completely occluding the single-channel pore, induces a subconducting state in which conductance is 20% that of the fully open channel. In a D172N mutant lacking the high-affinity polyamine-binding site, spermidine does not induce such a substate. However, the kinetics for the transitions between the substate and zero-current state in wild-type channels is the same as that of low-affinity block in the D172N mutant, supporting the notion that these are identical molecular events. Thus, the residual outward current after high-affinity spermidine block is susceptible to low-affinity block, which determines the final amplitude of the outward current. This study provides a detailed insight into the mechanism underlying the emergence of outward Kir2.1 currents regulated by inward rectification attributed to high- and low-affinity polyamine blocks.

Outward currents through inward rectifier K+ channels (Kir) play a pivotal role in determining resting membrane potential and in controlling excitability in many cell types. Thus, the regulation of outward Kir current (IK1) is important for appropriate physiological functions. It is known that outward IK1 increases with increasing extracellular K+ concentration ([K+]o), but the underlying mechanism is not fully understood. A "K+-activation of K+-channel" hypothesis and a "blocking-particle" model have been proposed to explain the [K+]o-dependence of outward IK1. Yet, these mechanisms have not been examined at the single-channel level. In the present study, we explored the mechanisms that determine the amplitudes of outward IK1 at constant driving forces [membrane potential (Vm) minus reversal potential (EK)]. We found that increases in [K+]o elevated the single-channel current to the same extent as macroscopic IK1 but did not affect the channel open probability at a constant driving force. In addition, spermine-binding kinetics remained unchanged when [K+]o ranged from 1 to 150 mM at a constant driving force. We suggest the regulation of K+ permeation by [K+]o as a new mechanism for the [K+]o-dependence of outward IK1.

This study demonstrates that carbon nanotubes (CNTs) can be fabricated into probes directly, with which neural activity can be monitored and elicited not only extracellularly but also intracellularly. Two types of CNT probes have been made and examined with the escape neural circuit of crayfish, Procambarus clarkia. The CNT probes are demonstrated to have comparable performance to conventional Ag/AgCl (silver/silver cloride) electrodes. Impedance measurement and cyclic voltammetry further indicate that the CNT probes transmit electrical signals through not only capacitive coupling but also resistive conduction. The resistive conduction facilitates the recording of postsynaptic potentials and equilibrium membrane potentials intracellularly as well as the delivery of direct-current stimulation. Furthermore, delivering current stimuli for a long term is found to enhance rather than to degrade the recording capability of the CNT probes. The mechanism of this fruitful result is carefully investigated and discussed. Therefore, our findings here support the suggestion that CNTs are suitable for making biocompatible, durable neural probes of various configurations for diverse applications.