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Studies on the Alterations of Expression and Function of Ion Channels in Diseases

Author: HanZhongYang
Tutor: WangXiaoLiang
School: Peking Union Medical College , China
Course: Molecular Pharmacology
Keywords: Pain Primary erythema acrodynia Sodium channel Nav1.7 SCN9A Ion channel disease Temperature Patch-clamp Transient receptor potential channel TRPM2 TRPM7 Cerebral ischemia Middle cerebral artery occlusion mRNA expression Real-time quantitative PCR Stably transfected Intracellular calcium Laser scanning confocal imaging technology
CLC: R363
Type: PhD thesis
Year: 2007
Downloads: 464
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Abstract

As the important transmembrane proteins, ion channels play a key role in the lifeof cells. And the alterations on the expressions and functions of ion channels havebeen found in lots of diseases. Meanwhile, ion channels are also the drug targets formany diseases. In the present thesis, we investigated the expressions and functions ofdifferent types of ion channels within two kinds of diseases, primary erythermalgiaand cerebral ischemia, in order to illuminate the roles of these channels in the relateddisorders.Part I Functional Studies of Ion Channels related with Primary ErythermalgiaPrimary erythermalgia (PEM) is a kind of autosomal dominant disease,characterized by intermittent burning pain with redness and heat in the extremities. Itcould be induced by heat or exercise, whereas keeping the involved extremities at anyicy cold temperature is the most effective way to relieve pain. It’s known little on thepathogenesis and the mechanism of PEM in the past. In recently, it’s reported that themutation of sodium channel Navl.7 could cause the syndromes of pain within PEM.In the present study, we carried on the electrophysiological studies of two newmutations which were found in Chinese PEM patients. Meanwhile, we investigatedthe physiological basis for the phenomenon that the pain within PEM can bealleviated by cooling.L858F mutation, a single amino acid substitution in Navl.7 was present in twochildren whose parents were asymptomatic. The asymptomatic father was geneticallymosaic for the mutation. The whole-cell patch clamp technique was used to comparethe electrophysiological characters of Nav1.7 wild-type channels and L858F mutants.Compared with wild-type, activation of L858F mutant channels was shifted by-9 mV,whereas steady-state inactivation was shifted by +3mV. There was a marked decreasein the rate of deactivation of L858F mutant channels, and the same change for the rate of closed-state inactivation. On the rate of recovery, L858F mutant channels showedthe significant faster than the wild-type channels. L858F also increased the rampcurrents which elicited by slow, small depolariztions, with 4 times larger than that ofwild-type channels. Our results suggested that L858F mutation could conferhyperexcitability on peripheral sensory neurons, and underlie PEM.Attacks of pain in PEM are alleviated by cooling of the limbs, but thephysiological basis for this phenomenon is not understood. Therefore, we investigatedthe influence of cooling on the biophysical properties of Nav1.7 wild-type and L858Fmutant channels. Whole-cell voltage-clamp measurements on wild-type or L858Fmutant channels expressed in HEK-293 cells revealed that cooling decreases currentdensity, slows deactivation and increases ramp currents for both wild-type and mutant.However, cooling differentially shifts the midpoint of steady-state activation in adepolarizing direction for L858F but not for wild type channels, which brings thethreshold of activation of the mutant channels closer to that of wild-type Navl.7 atlower temperatures. And we think that is likely to contribute to the alleviation ofpainful symptoms upon cooling in affected limbs of patient with PEM.V872G, a new mutation of Navl.7 was found in a Chinese girl with PEM. LikeL858F mutant, whole-cell patch clamp analysis was employed to characterizebiophysical properties of wild-type and the mutant channels in HEK-293 cells. Theresults showed that this new mutation produced a hyperpolarizing shift of about 3 mVin activation and a depolarizing shift of about 7 mV in steady-state inactivation. Alsolike L858F mutant, V872G mutant could decrease the rate of deactivationsignificantly and increase the rate of recovery. The rate of closed-state inactivation ofV872G was decreased significantly than that of wild-type. Meanwhile, the rampcurrent of V872G was about 2.5 times larger than that of wild-type. These changesshould increase excitability of nociceptive dorsal root ganglion neurons in which themutant channels are distributed, thus contributes to pain.In summary, we found that both L858F and V872G mutation present in patientswith PEM cause Nav1.7 ’gain of function’, which contributed to symptom productionin it. And the decreases in the difference of the activation between wild-type and mutants may contribute to the clinical observation that cooling alleviates pain.PartⅡGene Expressions of Transient Receptor Potential Channel Subtypes inRat Brain with Cerebral Ischemia & Relevant Pharmacological StudiesTransient Receptor Potential Channel (TRP channel), an important superfamilyof non-selective cation channels, with wide distribution and various functions, hasbeen one of the focuses in the research of ion channels recently.Stroke, including occlusive stroke, is one of the leading causes of death in theworld. It has been known that many pathophysiological mechanisms are responsiblefor the injury after cerebral ischemia and reperfusion, such as anoxia of neurons,bursting of free radicals and cumulation of acid substances.TRPM2 and TRPM7, two members of TRPM channel subfamily, have beendemonstrated to be regulated by anoxia and free radicals, and play an important rolein the death of neurons. In the present study, we used MCAO (middle cerebral arteryocclusion) to mimic the transient cerebral ischemia and permanent cerebral ischemia.And we observed the mRNA expressions ofTRPM2 & TRPM7 in rat brain cortex andhippocampus at different time points in these two pathophysiological conditions.Real-time PCR was employed to investigate the mRNA levels of TRPM2 &TRPM7 at 2h, 6h, 12h and 24h of reperfusion after 2h of MCAO during transientcerebral ischemia and the mRNA levels of TRPM2 & TRPM7 after 2h, 6h, 12h and24h of MCAO during permanent cerebral ischemia. In cortex, our results showed thatduring transient ischemia, the mRNA levels of TRPM2 was decreased by 66.3% at24h, and the mRNA levels of TRPM7 was decreased by 47.2%, 50.9% and 68.9% at2h, 12h and 24h respectively. And during permanent ischemia, the mRNA levels ofTRPM2 was decreased by 27.5%, 55.9% and 19.6% at 2h, 12h and 24h respectively,and the mRNA levels of TRPM7 was decreased by 26.6% and 34.4% at 2h and 24hrespectively. In hippocampus, we got the different results from the cortex. We foundthat during transient cerebral ischemia, there was not any alteration in the mRNAlevels of TRPM2 at these four time points, whereas the mRNA levels of TRPM7 was increased by 141.5% at 6h. Meanwhile, during permanent ischemia, the mRNA levelsof TRPM2 was increased by 47.5% and 31.3% at 2 h and 6h respectively, and themRNA levels of TRPM7 was increased by 1188.5% and 347.4% at 2h and 6h too.Our results suggested both TRPM2 and TRPM7 may play different roles duringthe ischemia of cortex and hippocampus, especially, TRPM7 may have importantroles during the ischemia of hippocampus.On the basis of the important physiological and pathophysiological functions ofTRPM7 channel, we constructed the stable cell lines transfected with TRPM7 gene.The expression of TRPM7 channel in the cell lines was regulated by tetracycline. Werecorded the characteristic TRPM7 channel currents in the cell lines after beingtreated with tetracycline and the amplitude of TRPM7 channel currents wasdependent on the dose and the treating-time of tetracycline. At the end, we studied theeffects of 1-NBP, a new anti-ischemia medicine, which invented by our research group,on the concentration of intracellular calcium with confocal assay. The results showedthat 1-NBP could decrease the concentration of intracellular calcium. And theseimplied that 1-NBP may have the inhibition effect on TRPM7 channel.

Studies on the Alterations of Expression and Function of Ion Channels in Diseases

Abstract

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