A B S T R A C T Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequencydependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley ntype gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.

Neurons with the capacity to discharge at high rates--'fast-spiking' (FS) neurons--are critical participants in central motor and sensory circuits. It is widely accepted that K+ channels with Kv3.1 or Kv3.2 subunits underlie fast, delayed-rectifier (DR) currents that endow neurons with this FS ability. Expression of these subunits in heterologous systems, however, yields channels that open at more depolarized potentials than do native Kv3 family channels, suggesting that they differ. One possibility is that native channels incorporate a subunit that modifies gating. Molecular, electrophysiological and pharmacological studies reported here suggest that a splice variant of the Kv3.4 subunit coassembles with Kv3.1 subunits in rat brain FS neurons. Coassembly enhances the spike repolarizing efficiency of the channels, thereby reducing spike duration and enabling higher repetitive spike rates. These results suggest that manipulation of K3.4 subunit expression could be a useful means of controlling the dynamic range of FS neurons.

[Arg 8 ]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca 2+ concentration (Ca 2+ spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K + (K v ) channels are crucial steps in this process. In the present study, K v currents (I Kv ) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I Kv . This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I Kv was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 μM) and XE-991 (10 μM) and enhanced by the KCNQ channel activator flupirtine (10 μM). BaCl 2 (100 μM) or linopirdine (5 μM) mimicked the effects of AVP on K + currents, AP generation, and Ca 2+ spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I Kv in A7r5 cells. I Kv was also inhibited in response to the PKC activator 4β-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I Kv by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca 2+ spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.Keywords potassium channel; signal transduction; membrane potential; calcium; vascular smooth muscle; M current Vasoconstrictor hormones cause contraction of vascular smooth muscle (VSM) cells by increasing cytosolic free Ca 2+ concentration ([Ca 2+ ] i ), which in turn activates the cells' contractile apparatus. Voltage-sensitive L-type Ca 2+ channels are known to be important in vasoconstrictor action (27), although the signaling pathways leading to activation of L-type channels are not well characterized. Influx of Ca 2+ via L-type channels is enhanced by NIH Public Access Author ManuscriptAm J Physiol Heart Circ Physiol. Author manuscript; available in PMC 2008 November 3. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript membrane depolarization, which may result from activation of nonselective cation currents (34,47,54) or Cl − currents (30). Alternatively, inhibition of outward K + currents could provide a depolarizing stimulus for activation of L-type Ca 2+ channels (38).We have previously demonstrated that concentrations of [Arg 8 ]-vasopressin (AVP) that may be found in the systemic circulation (10-500 pM) modulate the frequency of L-type Ca 2+ channel-dependent Ca 2+ spikes in A7r5 rat aortic smooth muscle cells. The stimulation of Ca 2+ -dependent action potentials, which underlie AVP-stimulated...

The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance.

A B S T R A C T The interaction of pancuronium with sodium channels was investigated in squid axons. Sodium current turns on normally but turns off more quickly than the control with pancuronium 0.1-1 mM present internally. The sodium tail current associated with repolarization exhibits an initial hook and then decays more slowly than the control. Pancuronium induces inactivation after the sodium inactivation has been removed by internal perfusion of pronase. Such pancuroniuminduced sodium inactivation follows a single exponential time course, suggesting first order kinetics which represents the interaction of the pancuronium molecule with the open sodium channel. The rate constant of association k with the binding site is independent of the membrane potential ranging from 0 to 80 mV, but increases with increasing internal concentration of pancuronium. However, the rate constant of dissociation 1 is independent of internal concentration of pancuronium but decreases with increasing the membrane potential. The voltage dependence of l is not affected by changing external sodium concentration, suggesting a current-independent conductance block. The steady-state block depends on the membrane potential, being more pronounced with increasing depolarization, and is accounted for in terms of the voltage dependence of l. A kinetic model, based on the experimental observations and the assumption on binding kinetics of pancuronium with the open sodium channel, successfully simulates many features of sodium current in the presence of pancuronium. I N T R O D U C T I O NM e m b r a n e ionic channels can be subject to pharmacological, chemical, and enzymatic modifications. T h e inactivation process of the Na-conducting system is selectively modified by the enzyme pronase ( A r m s t r o n g et al., 1973) or by the specific protein reagent N -b r o m o a c e t a m i d e (Oxford et al., 1976), yet the activation process is not affected by either of them. This suggests different identities of the macromolecules responsible for activation and inactivation mechanisms.T h e direct interaction of chemicals with o p e n sodium channels has been p r o p o s e d to explain the voltage-dependent block of sodium conductance by H ÷ and Ca ++ ions (Woodhull, 1973), the frequency-and voltage-dependent block of sodium conductance by lidocaine derivatives (Strichartz, 1973;Courtney, 1975;Hille et al., 1975) 9-aminoacridine (Yeh and Narahashi, 1975b) a n d strychnine