Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4β is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2 ). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50 , the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the βancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 μM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin- L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM Nlobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu- 593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50 , the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the βancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 μM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin- L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM Nlobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu- 593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Persistent Erectile Dysfunction after Discontinuation of 5-Alpha Reductase Inhibitor Therapy in Rats Depending on the Duration of Treatment

PURPOSE: The current study is aimed to assess whether a longer duration of 5α-reductase inhibitor (5α-RI) exposure was associated with higher rate of permanent erectile dysfunction (ED) in a rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats (n=76) were assigned to five groups: (i) normal control group; (ii) dutasteride (0.5 mg/rat/d) for 4-weeks group; (iii) dutasteride for 4-weeks plus 2-weeks of resting group; (iv) dutasteride for 8-weeks group; and (v) dutasteride for 8-weeks plus 2-weeks of resting group. In vivo erectile responses to electrical stimulation, and changes of fibrotic factors and smooth muscle/collagen contents in the corpus cavernosum were evaluated in each group. RESULTS: Dutasteride administration for 4 and 8 weeks significantly decreased erectile parameters compared with the control group. Reduced erectile responses were recovered during 2 weeks of drug-free time in the 4-week treatment group, but were not in the 8-week group. Protein levels of fibrosis-related factors transforming growth factor (TGF)-β1, TGF-β2, and p-Smad/Smad (Smad 2/3) in the corpus cavernosum showed no significant change after 4 weeks of dutasteride oral administration, but were enhanced after 8 weeks. Dutasteride markedly decreased smooth muscle content and increased collagen after 4 and 8 weeks of use, but no nuclear size changes; however, neither group showed significant improvement in the smooth muscle to collagen ratio after the rest period. CONCLUSIONS: Our study showed that recovery from ED depended on the duration of medication, and administration of dutasteride for more than 8-weeks in rats could result in irreversible ED even after discontinuation of medication.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

The role of calmodulin in regulating calcium-permeable PKD2L1 channel activity

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (–)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.

Electrophysiological characteristics of R47W and A298T mutations in CLC-1 of myotonia congenita patients and evaluation of clinical features

Myotonia congenita (MC) is a genetic disease that displays impaired relaxation of skeletal muscle and muscle hypertrophy. This disease is mainly caused by mutations of CLCN1 that encodes human skeletal muscle chloride channel (CLC-1). CLC-1 is a voltage gated chloride channel that activates upon depolarizing potentials and play a major role in stabilization of resting membrane potentials in skeletal muscle. In this study, we report 4 unrelated Korean patients diagnosed with myotonia congenita and their clinical features. Sequence analysis of all coding regions of the patients was performed and mutation, R47W and A298T, was commonly identified. The patients commonly displayed transient muscle weakness and only one patient was diagnosed with autosomal dominant type of myotonia congenita. To investigate the pathological role of the mutation, electrophysiological analysis was also performed in HEK 293 cells transiently expressing homo- or heterodimeric mutant channels. The mutant channels displayed reduced chloride current density and altered channel gating. However, the effect of A298T on channel gating was reduced with the presence of R47W in the same allele. This analysis suggests that impaired CLC-1 channel function can cause myotonia congenita and that R47W has a protective effect on A298T in relation to channel gating. Our results provide clinical features of Korean myotonia congenita patients who have the heterozygous mutation and reveal underlying pathophyological consequences of the mutants by taking electrophysiological approach.

The Pathophysiologic Roles of TRPM7 Channel

Transient receptor potential melastatin 7 (TRPM7) is a member of the melastatin-related subfamily and contains a channel and a kinase domain. TRPM7 is known to be associated with cell proliferation, survival, and development. It is ubiquitously expressed, highly permeable to Mg2+ and Ca2+, and its channel activity is negatively regulated by free Mg2+ and Mg-complexed nucleotides. Recent studies have investigated the relationships between TRPM7 and a number of diseases. TRPM7 regulates cell proliferation in several cancers, and is associated with ischemic cell death and vascular smooth muscle cell (VSMC) function. This review discusses the physiologic and pathophysiologic functions and significance of TRPM7 in several diseases.

Effects of Histamine on Cultured Interstitial Cells of Cajal in Murine Small Intestine

Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external Ca2+ influx and Ca2+ release from internal stores in a PLC and PLD dependent manner.

Functional Analysis of TRPV6 Polymorphisms / 한국실험동물학회지

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel transient receptor potential vanilloid 6 (TRPV6). The putatively-selected TRPV6 haplotype contains three candidate sites for functional differences, namely derived non-synonymous substitutions C157R, M378V and M681T. Functional electrophysiological characteristics between wild-type and mutant (C157R, M378V and M681T) TRPV6 proteins were investigated by cloning the mutant TRPV6 forms, transfecting cell lines, and carrying out electrophysiology experiments via patch clamp analysis. No statistically significant differences in biophysical channel function were found although one property, namely Ca2+-dependent inactivation, may show functionally-relevant differences between the wild-type and mutant TRPV6 proteins. This study shows that Ca2+-dependent inactivation is one of the good differentiation characteristics in TRPV6, and will be useful in an advancing our knowledge about TRPV6.

The Role of Capsaicin in Spontaneous Pacemaking Activity in Gastrointestinal Tract

No abstract available.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba2+ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K+ currents in the absence and presence of externally applied Ba2+. Wild type Kir2.1 was blocked by externally applied Ba2+ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba2+ blockage. The estimated Kd(0) was 108micrometer for Kir2.1 wild type, 124micrometer for a concatameric WT-Y145V dimer, 109micrometer for a WT-Y145L dimer, and 267micrometer for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba2+, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba2+ and a decrease in the on transition rate constant (kon). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba2+ blockage in Kir 2.1.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 micrometer filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.

Inhibition of the Desensitization of Canonical Transient Receptor Potential Channel 5 by Dimethyl Sulfoxide

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca2+-permeable cation channel in mammalian cells. TRPC5 is rapidly desensitized after activation by G protein-coupled receptor. Herein we report the effect of dimethyl sulfoxide (DMSO) on the desensitization of TRPC5. TRPC5 was initially activated by muscarinic stimulation with 50microM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). DMSO in the pipette solution slowed the rate of this desensitization. Under the control conditions, TRPC5 current spontaneously declined to 6+/-1% of the initial peak amplitude 60 sec after CCh application and to 1+/-0.5% after 120 sec. But, in the presence of 0.01%, 0.1% and 1% DMSO, TRPC5 current spontaneously declined to 55+/-2%, 68+/-1% and 100+/-0.2% of the initial peak amplitude 60 sec after CCh application and to 38+/-2%, 61+/-1% and 100+/-1% after 120 sec, respectively. The results suggest that DMSO can internally attenuate the desensitization of TRPC5 current through unknown mechanisms that remain to be elucidated.

Methylene Blue-stained Interstitial Cells are Electrically Active in the Myenteric Board Freshly Prepared from the Murine Small Intestine

Many gastrointestinal muscles show electrical oscillation, so-called "slow wave", originated from interstitial cells of Cajal (ICCs). Thus, a technique to freshly isolate the cells is indispensable to explore the electrophysiological properties of the ICCs. To apply an enzyme solution on the serosal surface for cell isolation, the intestine was inverted and 0.02% trypsin solution and 0.04% collagenase solution were applied to serosal cavity. After the enzyme treatment, mucosal layer was removed and longitudinal muscle layer was gently separated from the rest of tissue. The thin layer was stretched in the recording chamber and mounted on an inverted microscope. Using beta-escine, perforated whole cell patch clamp technique was used. Under a microscope, the tissue showed smooth muscle cells and interstitial cells around the myenteric plexus. Under voltage clamp condition, three types of membrane potential were recorded. One group of interstitial cells, which were positive to methylene blue and CD34, showed spontaneous outward current. These cells had bipolar shape and were considered as fibroblast-like cells because of their peculiar shape and arrangement. Another group, positive to c-kit and methylene blue, showed spontaneous inward current. These cells had more rounded shape and processes and were considered as ICCs. The third, positive to c-kit and had granules containing methylene blue, showed quiet membrane potentials under the voltage-clamp mode. These cells appeared to be resident macrophages. Therefore, in the freshly isolated thin tissue preparation, methylene blue could easily identify three types of cells rather than morphological properties. Using this method, we were able to study electrical properties of fibroblast and residential macrophage as well as myenteric ICCs.