Xiandi Gong

Stroke therapy in traditional Chinese medicine (TCM): prospects for drug discovery and development

Brain injuries resulting from stroke are a major and increasing public health problem in both developed and developing countries worldwide. Chinas extensive experience in the use of traditional Chinese medicines (TCMs) in stroke therapy indicates that TCM preparations are effective, with few or no side-effects. There are more than 100 traditional medicines in use for stroke therapy in China. Some of their therapeutic effects in stroke have been confirmed by recent clinical studies. A large number of compounds have been isolated from TCMs and most of these resources have not yet been characterized for pharmacological purposes. Here, this article explains how TCM provides an extensive and knowledge-rich foundation for implementing a strategically focused pharmacological research programme aimed at the development of new drugs.

Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.

Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)2− anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 μm Au(CN)2−, a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Relative Au(CN)2− permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337‐S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent.

Direct Comparison of the Functional Roles Played by Different Transmembrane Regions in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, little is known about the relative functional contribution of different TM regions to the pore. We have used patch clamp recording to investigate the functional consequences of point mutations throughout the six transmembrane regions in the N-terminal part of the CFTR protein (TM1-TM6). A range of specific functional assays compared the single channel conductance, anion binding, and anion selectivity properties of different channel variants. Overall, our results suggest that TM1 and -6 play dominant roles in forming the channel pore and determining its functional properties, with TM5 perhaps playing a lesser role. In contrast, TM2, -3, and -4 appear to play only minor supporting roles. These results define transmembrane regions 1 and 6 as major contributors to the CFTR channel pore and have strong implications for emerging structural models of CFTR and related ATP-binding cassette proteins.

Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2− ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2− binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non‐charge‐dependent effects also appear to contribute to Au(CN)2− binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2− ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion‐ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism.

Mutation-induced Blocker Permeability and Multiion Block of the CFTR Chloride Channel Pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)4 2− acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent K d 556 μM), kinetically fast, and weakened by extracellular Cl− ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)4 2− ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)4 2− increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)4 2− ions to the extracellular solution. This “punchthrough” was prevented by extracellular Cl− ions, reminiscent of a “lock-in” effect. Relief of block in F337A by Pt(NO2)4 2− permeation was only observed for blocker concentrations above 300 μM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl− current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)4 2− in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)4 2− to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)4 2− ions within the pore promotes their permeation to the extracellular solution.

N-Methyl-D-Aspartate Receptor Antagonist Activity in Traditional Chinese Stroke Medicines

Traditional Chinese medicine (TCM) has a long history in stroke therapy and its therapeutic efficacy has been confirmed by clinical studies. The molecular basis of the neuroprotective effects is unknown. We wondered whether or not the neuroprotective effect of TCMs might be due to their N-methyl-D-aspartate (NMDA) receptor (NMDAR) antagonist properties. We used the patch-clamp technique to screen 22 TCM stroke drugs for NMDAR antagonist activity in cultured cortical neurons. The drugs were also screened for their ability to abate NMDA-induced neurotoxicity. Aqueous extracts of Scutellaria baicalensis, Stephania tetrandra, and Salvia miltiorrhiza blocked currents induced by NMDA (200 µM, 10 µM glycine, 0 Mg2+) at a holding potential of –80 mV by 83.45 ± 4.34, 38.65 ± 7.50, and 52.97 ± 1.78%, respectively. The block of the NMDA-evoked currents was voltage-dependent and showed a negative slope conductance reminiscent of Mg2+. Atomic absorption spectrophotometry revealed the presence of 12.5, 2, and 8.7 mM Mg2+ in the extracts of S. baicalensis,S. tetrandra, and S. miltiorrhiza, respectively. None of these extracts blocked NMDA-induced neuronal death. The Uncaria rhynchophylla extract blocked NMDA-evoked currents by 54.98 ± 8.61% even at +60 mV and reduced NMDA-induced neuronal death by 59.13 ± 3.52%. NMDAR antagonist activity may underlie the neuroprotective effects of this TCM. Some TCM drugs may exert therapeutic effects due to their Mg2+ content.

Formation of the Epididymal Fluid Microenvironment

It was thirty years ago when it was demonstrated that spermatozoa have to go through maturational changes in the epididymis before they can fertilize the ovum (Orgebin-Crist, 1967; Bedford, 1967). Since then, there has been a growing focus on the physiology and biochemistry of the epididymis. This burst of epididymal research was largely driven by the recognition that contraceptive agents that interfere with sperm maturation in the epididymis have many advantages over those that suppress sperm production in the testis. The former agents are expected to have a quick onset of action, rapid reversibility upon withdrawal, and reduced chance of mutagenic damage and endocrine impairment of libido. These advantages have been borne out by α-chlorohydrin (Coppola, 1969) and the chlorinated sugars (Ford and Waites, 1980) which act by interfering with sperm metabolism in the epididymis. However, interest in these agents as potential male contraceptives soon waned due to their neurotoxicity. In recent years, the advent of molecular biology techniques has led to t he identification and cloning of genes encoding sperm-coating or epididymal-specific proteins which can be targeted for immunocontraception. However, for these methods to be effective, organ-specific delivery methods of antisense oligonucleotides/antibodies will have to be developed. Reduction of the prominent constituents of the epididymal fluid viz. α-glucosidase and L-carnitine or enhancement of sperm transport through the epididymis by pharmacological means has not led to infertility in animals (Cooper and Yeung, 1999). Despite the consensus held that the epididymis is indispensable for the full expression of male fertility, attempts to induce infertility via an epididymal approach remain elusive.

Multiple inhibitory effects of Au(CN)2− ions on cystic fibrosis transmembrane conductance regulator Cl− channel currents

Lyotropic pseudohalide anions are potentially useful as high affinity probes of Cl− channel pores. However, the interaction between these pseudohalides and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel have not been described in detail. Here we show that Au(CN)2− ions applied to the intracellular face of membrane patches from stably transfected baby hamster kidney cells inhibit CFTR channel currents by at least two mechanisms, which can be distinguished at the single channel level or by inhibiting channel closure using 2 mm pyrophosphate. Low concentrations (< 10 μm) of Au(CN)2− significantly reduced CFTR channel open probability. This effect was apparently voltage insensitive, independent of extracellular Cl− concentration, and lost following exposure to pyrophosphate. Higher concentrations of intracellular Au(CN)2− caused an apparent reduction in unitary current amplitude, presumably due to a kinetically fast blocking reaction. This effect, isolated following exposure to pyrophosphate, was strongly voltage dependent (apparent Kd 61.6 μm at −100 mV and 913 μm at +60 mV). Both the affinity and voltage dependence of block were highly sensitive to extracellular Cl− concentration. We propose that Au(CN)2− has at least two inhibitory effects on CFTR currents: a high affinity effect on channel gating due to action on a cytoplasmically accessible aspect of the channel and a lower affinity block within the open channel pore. These results offer important caveats for the use of lyotropic pseudohalide anions such as Au(CN)2− as specific high affinity probes of Cl− channel pores.

Mechanism of lonidamine inhibition of the CFTR chloride channel

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage‐dependent block of CFTR currents, with an apparent Kd of 58 μM at −100 mV. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl− concentration. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 μM−1 s−1 and an off‐rate of 143 s−1. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action.

Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Rat Efferent Duct Epithelium

Abstract The expression of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in rat efferent ducts. Under whole-cell patch-clamp condition, efferent duct cells responded to intracellular cAMP with a rise in inward current. The cAMP-activated current exhibited a linear I–V relationship and time- and voltage-independent characteristics. The current was inhibited by the Cl− channel blocker diphenylamine 2,2′-dicarboxylic acid (DPC) in a voltage-dependent manner and reversed at 24 ± 0.5 mV, close to the equilibrium potential for Cl− (30 mV), suggesting that the current was Cl− selective. The cAMP-activated current displayed a permeability sequence of Br− > Cl− > I−. Short-circuit current measurement in cultured rat efferent duct epithelia also revealed a cAMP-activated inward current inhibitable by DPC. These electrophysiological properties of the cAMP-activated Cl− conductance in the efferent duct were consistent with those reported for CFTR. In support of the functional studies, reverse transcription polymerase chain reaction revealed the presence of CFTR message in cultured efferent duct epithelium. Immunohistochemical studies in intact rats also demonstrated CFTR protein at the apical membrane of the principal cells of efferent duct. CFTR may play a role in modulating fluid transport in the efferent duct.