Revisiting inward rectification: K ions permeate through Kir2.1 channels during high-affinity block by spermidine.

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 Mg(2+) 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.

Extracellular K+ elevates outward currents through Kir2.1 channels by increasing single-channel conductance.

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.

The extracellular K+ concentration dependence of outward currents through Kir2.1 channels is regulated by extracellular Na+ and Ca2+.

It has been known for more than three decades that outward Kir currents (I(K1)) increase with increasing extracellular K(+) concentration ([K(+)](o)). Although this increase in I(K1) can have significant impacts under pathophysiological cardiac conditions, where [K(+)](o) can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K(+)](o) dependence of Kir2.1-mediated outward I(K1) was due to [K(+)](o)-dependent inhibition of outward I(K1) by extracellular Na(+) and Ca(2+). This could be accounted for by Na(+)/Ca(2+) inhibition of I(K1) through screening of local negative surface charges. Consistent with this, extracellular Na(+) and Ca(2+) reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I(K1) in a similar [K(+)](o)-dependent manner as Na(+)/Ca(2+). Site-directed mutagenesis studies identified Asp(114) and Glu(153) as the source of surface charges. Reducing K(+) activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na(+) and Ca(2+), suggesting that in addition to exerting a surface electrostatic effect, Na(+) and Ca(2+) might inhibit outward I(K1) by inhibiting K(+) activation. This study identified interactions of K(+) with Na(+) and Ca(2+) that are important for the [K(+)](o) dependence of Kir2.1-mediated outward I(K1).

Interfacing neurons both extracellularly and intracellularly using carbon-nanotube probes with long-term endurance.

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.