Amphiphilic blockers punch through a mutant CLC-0 pore.

Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl(-), the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than -80 mV, a phenomenon similar to the blocker "punch-through" shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at -80 mV in symmetrical 140 mM Cl(-), and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl(-) reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl(-) reversal potential. That the shift of the CPA-dissociation rate follows the Cl(-) gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl(-) reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement

Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Members of the CLC family of Cl(-) channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl(-) channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd(2+)-coordinating residues. The inhibition of the current of CLC-0 by Cd(2+) was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd(2+)-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd(2+)-binding site. Kinetic studies showed that Cd(2+) inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd(2+) modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl(-) channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions

Analytical result on the supercurrent through a superconductor/quantum-dot/superconductor junction

We present an analytical result for the supercurrent across a superconductor/quantum-dot/superconductor junction. By converting the current integration into a special contour integral, we can express the current as a sum of the residues of poles. These poles are real and give a natural definition of the Andreev bound states. We also use the exact result to explain some features of the supercurrent transport behavior.Comment: 8 pages, 2 figure

Andreev reflection through a quantum dot coupled with two ferromagnets and a superconductor

We study the Andreev reflection (AR) in a three terminal mesoscopic hybrid system, in which two ferromagnets (F$_1$ and F$_2$) are coupled to a superconductor (S) through a quantum dot (QD). By using non-equilibrium Green function, we derive a general current formula which allows arbitrary spin polarizations, magnetization orientations and bias voltages in F$_1$ and F$_2$. The formula is applied to study both zero bias conductance and finite bias current. The current conducted by crossed AR involving F$_1$, F$_2$ and S is particularly unusual, in which an electron with spin $\sigma$ incident from one of the ferromagnets picks up another electron with spin $\bar{\sigma}$ from the other one, both enter S and form a Cooper pair. Several special cases are investigated to reveal the properties of AR in this system.Comment: 15 pages, 7 figures, LaTe