Biomedical subjects

Chun Jiang

Publications and source records attributed to Chun Jiang.

At least 19 recordsLinked to original sources

PubMed2006

High CO2 chemosensitivity versus wide sensing spectrum: a paradoxical problem and its solutions in cultured brainstem neurons.

CO2 central chemoreceptors play an important role in cardiorespiratory control. They are highly sensitive to P(CO2) in a broad range. These two sensing properties seem paradoxical as none of the known pH-sensing molecules can achieve both. Here we show that cultured neuronal networks are likely to solve the sensitivity versus spectrum problem with parallel and serial processes. Studies were performed on dissociated brainstem neurons cultured on microelectrode arrays. Recordings started after a 3 week initial period of culture. A group of neurons were dose-dependently stimulated by elevated CO2 with a linear response ranging from 20 to 70 Torr. The firing rate of some neurons increased by up to 30% in response to a 1 Torr P(CO2) change, indicating that cultured brainstem neuronal networks retain high CO2 sensitivity in a broad range. Inhibition of Kir channels selectively suppressed neuronal responses to hypocapnia and mild hypercapnia. Blockade of TASK channels affected neuronal response to more severe hypercapnia. These were consistent with the pKa values measured for these K+ channels in a heterologous expression system. The CO2 chemosensitivity was reduced but not eliminated by blockade of presynaptic input from serotonin, substance P or glutamate neurons, indicating that both pre and postsynaptic neurons contribute to the CO2 chemosensitivity. These results therefore strongly suggest that the physiological P(CO2) range appears to be covered by multiple sensing molecules, and that the high sensitivity may be achieved by cellular mechanisms via synaptic amplification in cultured brainstem neurons.

Animals↗

PubMed2006

Gating of the ATP-sensitive K+ channel by a pore-lining phenylalanine residue.

ATP-sensitive K(+) (K(ATP)) channels are gated by intracellular ATP, proton and phospholipids. The pore-forming Kir6.2 subunit has all essential machineries for channel gating by these ligands. It is known that channel gating involves the inner helix bundle of crossing in which a phenylalanine residue (Phe168) is found in the TM2 at the narrowest region of the ion-conduction pathway in the Kir6.2. Here we present evidence that Phe168-Kir6.2 functions as an ATP- and proton-activated gate via steric hindrance and hydrophobic interactions. Site-specific mutations of Phe168 to a small amino acid resulted in losses of the ATP- and proton-dependent gating, whereas the channel gating was well maintained after mutation to a bulky tryptophan, supporting the steric hindrance effect. The steric hindrance effect, though necessary, was insufficient for the gating, as mutating Phe168 to a bulky hydrophilic residue severely compromised the channel gating. Single-channel kinetics of the F168W mutant resembled the wild-type channel. Small residues increased P(open), and displayed long-lasting closures and long-lasting openings. Kinetic modeling showed that these resulted from stabilization of the channel to open and long-lived closed states, suggesting that a bulky and hydrophobic residue may lower the energy barrier for the switch between channel openings and closures. Thus, it is likely that the Phe168 acts as not only a steric hindrance gate but also potentially a facilitator of gating transitions in the Kir6.2 channel.

Adenosine Triphosphate↗

PubMed2006

Multicellular recordings of cultured brainstem neurons in microelectrode arrays.

Several vital systemic functions are controlled by the brainstem, which has been studied in a variety of experimental preparations and by various techniques, including in-vitro electrophysiological preparations. Although these in-vitro approaches have greatly advanced the understanding of brainstem neurons, most recording methods with microelectrodes and patch pipettes are invasive. To take advantage of in-vitro approaches but avoid their potential problems, we have studied brainstem neurons in microelectrode arrays (MEA). Neurons were isolated from the medulla oblongata and cultured in DMEM. Extracellular recordings were performed with no evident perturbations to the cellular environment. Neurons started firing after 24-48 h in culture, reached stable activity in 3-4 weeks, and retained this activity for at least 3 months. From their firing patterns, these neurons could be divided into tonic and bursting units. The latter could be further divided into regular and irregular bursters based on their burst intervals. Cells were stimulated or inhibited by exposure to 10% CO2. The stimulatory effect of CO2, though smaller, was still seen after selective ablation of serotonergic neurons or with low Ca++ and high Mg++ in the extracellular medium. Similar treatments had no significant effect on CO2-inhibited units. The abundance of units with respect to their firing patterns and CO2 responses, together with the long-term stable non-invasive recordings with no evident perturbation to cellular environments, suggests that MEA represent another promising in-vitro approach for studying brainstem neurons.

Animals↗

PubMed2006

Elimination of allosteric modulation of myocardial KATP channels by ATP and protons in two Kir6.2 polymorphisms found in sudden cardiac death.

The major cause of sudden cardiac death (SCD) is ventricular arrhythmias due to unstable myocardial electrical activity in which the ATP-sensitive K+ (KATP) channels play a role. Genetic disruption of these channels predisposes the myocardium to arrhythmias. Two point mutations in the Kir6.2 subunit are found in SCD with acute myocardial infarction. Here we show evidence for the functional consequences of the P266T and R371H variants. Baseline single-channel properties, expression density, and channel modulations were studied in patch clamp. We focused on channel modulations by intracellular ATP and protons, as the concentration of these two important KATP channel regulators changes widely with hypoxic ischemia. We found that both variants expressed functional currents even though they occur at two highly conserved regions. The open state probability of P266T was twice as high as the wild-type (WT) channel, whereas its channel density was only approximately 20% of the WT channel. Although the outward current was not affected by these two mutations at neutral pH, it was approximately 20% lower at acidic pH in the P266T than in the WT channel. Both P266T and R371H mutations significantly reduced ATP sensitivity and increased pH sensitivity. More dramatically, allosteric regulation by intracellular ATP and protons was almost completely eliminated in the polymorphic P266T and R371H channels. Such an abnormality was seen in both inward and outward currents. Given the importance and beneficial effects of allosteric regulation in cellular responses to metabolic stress, the loss of such a regulatory mechanism in the P266T and R371H variants appears consistent with the adverse consequences occurring during acute myocardial infarction in patients.

ATP-Binding Cassette Transporters↗

PubMed2005

CO2 central chemosensitivity: why are there so many sensing molecules?

CO2 central chemoreceptors (CCRs) play a critical role in respiratory and cardiovascular controls. Although the primary sensory cells and their neuronal networks remain elusive, recent studies have begun to shed insight into the molecular mechanisms of several pH sensitive proteins. These putative CO2/pH-sensing molecules are expressed in the brainstem, detect P(CO2) at physiological levels, and couple the P(CO2) to membrane excitability. Functional analysis suggests that multiple CO2/pH-sensing molecules are needed to achieve high sensitivity and broad bandwidth of the CCRs. In contrast to the diversity of pH sensitive molecules, molecular mechanisms for CO2 sensing are rather general. The sensing molecules detect pH changes rather than molecular CO2. One or a few titratable amino acid residues in these proteins are usually involved. Protonation of these residues may lead to a change in protein conformation that is coupled to a change in channel activity. Depending on the location of the protonation sites, a membrane protein can detect extra- and/or intracellular pH.

Animals↗

PubMed2005

Subunit stoichiometry of the Kir1.1 channel in proton-dependent gating.

Kir1.1 channel regulates membrane potential and K+ secretion in renal tubular cells. This channel is gated by intracellular protons, in which a lysine residue (Lys80) plays a critical role. Mutation of the Lys80 to a methionine (K80M) disrupts pH-dependent channel gating. To understand how an individual subunit in a tetrameric channel is involved in pH-dependent channel gating, we performed these studies by introducing K80M-disrupted subunits to tandem tetrameric channels. The pH sensitivity was studied in whole-cell voltage clamp and inside-out patches. Homomeric tetramers of the wild-type (wt) and K80M-disrupted channels showed a pH sensitivity almost identical to that of their monomeric counterparts. In heteromeric tetramers and dimers, pH sensitivity was a function of the number of wt subunits. Recruitment of the first single wt subunit shifts the pK(a) greatly, whereas additions of any extra wt subunit had smaller effects. Single-channel analysis revealed that the tetrameric channel with two or more wt subunits showed one substate conductance at approximately 40% of the full conductance, suggesting that four subunits act as two pairs. However, three and four substates of conductance were seen in the tetrameric wt-3K80M and 4K80M channels. Acidic pH increased long-time closures when there were two or more wt subunits. Disruption of more than two subunits led to flicking activity with appearance of a new opening event and loss of the long period of closures. Interestingly, the channel with two wt subunits at diagonal and adjacent configurations showed the same pH sensitivity, substate conductance, and long-time closure. These results thus suggest that one functional subunit is sufficient to act in the pH-dependent gating of the Kir1.1 channel, the channel sensitivity to pH increases with additional subunits, the full pH sensitivity requires contributions of all four subunits, and two subunits may be coordinated in functional dimers of either trans or cis configuration.

Animals↗

PubMed2005

Laparoscopic radical cystectomy with orthotopic ileal neobladder: report of 33 cases.

BACKGROUND: The laparoscopic radical cystectomy (LRC) with orthotopic ileal neobladder is now applied to treat invasive bladder cancer, however, it has not been well codified and illustrated. We describe in this paper a technique step by step that we have developed in 33 patients and achieved excellent results. METHODS: The surgical procedure can be divided into eight steps: laparoscopic pelvic lymphadenectomy and mobilization of the distal ureters; exposing Denonvillier's space and the posterior aspect of prostate; exposing retropubic space and anterior surface of the bladder; dividing the lateral pedicles of the bladder and the prostate; dividing the apex of the prostate; extracorporeal formation of the ileal pouch; extracorporeal implantation of the ureters; and laparoscopic urethra-neobladder anastomosis. This operation was performed in 33 patients, 29 males and 4 females, with muscle invasive bladder cancer between December 2002 and September 2004. RESULTS: The operating time was 5.5-8.5 hours with an average of 6.5 hours; the estimated blood loss was 200-1000 ml with an average of 460 ml. The surgical margins of the bladder specimen were negative in all patients. There was no evidence of local recurrence at follow-up of 1-21 months in all the patients. However lymph node metastases were found in one case at 9 months postoperatively. Most of patients achieved urine control 1 to 3 months after surgery. The daytime continence rate was 94% (31 cases) and nighttime continence rate was 88% (29 cases). Urodynamic evaluation was performed between 3 and 6 months postoperatively for all cases. The mean value of neobladder capacity was (296 +/- 37) ml. The mean value of maximum flow rate was (18.7 +/- 7.1) ml/s. The mean residual urine volume was (32 +/- 19) ml. In all cases, excretory urography at 1 to 2 months postoperatively demonstrated slightly dilated upper urinary tracts without ureteral obstruction, which resolved at follow up. Cystography showed neobladders being similar in shapes to normal. Two small ureteral nipples with intermittently efflux of urine were observed at cystoscopy in most patients. Postoperative complications occurred in 6 of 33 patients (18%), including pouch leakage in 2 cases, pelvic infection in 1, partial small bowel obstruction in 2 and neobladder-vaginal fistula in 1. CONCLUSIONS: The LRC with orthotopic ileal neobladder is a feasible option for bladder cancer when radical cystectomy is indicated. The extracorporeal formation of the ileal pouch and ureteral implantation through a small lower midline incision can simplify the complexity of the procedures, shorten the duration of surgery and reduce the medical expenses.

Adult↗

PubMed2005

Single nucleotide polymorphisms in K(ATP) channels: muscular impact on type 2 diabetes.

ATP-sensitive K+ channels (K(ATP) channels) play an important role in glucose homeostasis. A single nucleotide polymorphism (SNP) in the Kir6.2 subunit causes a point mutation of Glu23 to lysine and reduces the ATP sensitivity of pancreatic K(ATP) channels. The SNP found in 58% of Caucasians accounts for 15% of type 2 diabetes. Here we show evidence for dysregulations of muscular K(ATP) channels with the E23K variation. We were particularly interested in the channel modulation by intracellular protons, as pH changes widely and frequently in skeletal muscles. Surprisingly, we found that the defect of the E23K variant was more related to pH than ATP. A level of intracellular acidification seen during exercise not only activated the E23K channel more readily than the wild type, but also relieved the channel inhibition by ATP, leading to a vast increase in the channel open-state probability by approximately sevenfold at pH 6.8 over the wild-type channel at pH 7.4. Considering the reduction in sarcolemmal excitability, muscle fatigue, and impairment of muscular glucose uptake found previously by genetically disrupting K(ATP) channels, it is likely that the E23K variant in muscular K(ATP) channels affects systemic glucose homeostasis and poses an important risk factor for type 2 diabetes and obesity.

Amino Acid Substitution↗

PubMed2005

[Identification of response element gene sequence for non-steroid hormone transcription factors for the activation and up-regulation of L-plastin expression in prostate cancer].

OBJECTIVE: To search and identify the non-steroid receptor binding cis-acting elements in the L-plastin promoter in prostate cancer, and the correlative regulation pathway and transcription factors. METHODS: On the basis of construction of the L-plastin promoter luciferase vectors which were removed the steroid hormone receptor AR and ER binding elements, the promoter on the vector was nest-deleted by Exonuclease III and the relative luciferase plasmids were constructed. Transfected these twelve plasmids into prostate cancer cell line LNCaP under dihydrotestosterone-stimulated situation or not and test the intensity of luciferase, then we got the regulation message of every 200 bp part of the promoter in prostate cancer. After the analysis of relative programme, we got the possible regu- lation pathway of non-steroid hormone transcription factors. After removing the possible transcription factors binding site sequence by site-specific mutagenesis, the changes luciferase of activities proved our reasoning. RESULTS: We succeed in segmental deletion of the L-plastin promoter, and constructing the relative plasmids containing part L-plastin promoter on luciferase vector pGL3-basic. After testing the luciferase activities of constructed plasmids, we found the sequence from 206 to 1 of L-plastin promoter had significant luciferase activity. The software TRANSFECT showed that there were binding elements for transcription factors AP-4 at seq-198 to 192 and SP-1 at seq-54 to 41 on the short part promoter (206 to 1). The recombinant plasmids deleted the AP-4 and SP-1 binding elements had lower luciferase activity than the wild-type. CONCLUSION: There are some other non-steroid hormone pathway to regulate the expression of L-plastin except the steroid hormone pathway in prostate cancer. The main binding sites of the non-steroid hormone regulator lies in the sequence from 206 to 1. Transcription factors AP4 and SP-1 may up-regulated the expression of L-plastin by binding these sites.

Animals↗

PubMed2005

[Needle laparoscopic varicocelectomy].

OBJECTIVE: We evaluated our method and effects of needle laparoscopic varicocelectomy for the treatment of varicocele. METHODS: 72 patients (105 lateral) diagnosed varicocele were performed laparoscopic varicocelectomy under epidural combined intravenous anesthesia from Feb, 2003 to Apr, 2005. Two 2 mm incisions and one 5 mm incision were made on the midline of lower abdomen, by which two 2 mm trocars and one 5 mm trocar were introduced. Vessel-sealing device (Ligasure) was used to seal the internal spermatic veins. RESULTS: All operations were completed successfully. Mean operation time was 15 minutes unilateral and 21 minutes bilateral. The patients were hospitalized for 3 to 5 days after procedure. Follow-up was scheduled for 6 to 12 months and there was no recurrence. CONCLUSION: Needle laparoscopic varicocelectomy gives favorable effect with minimal invasion, rapid recovery, which is the best choice for the treatment of varicocele.

Adolescent↗

PubMed2004

Disruption of glucose sensing and insulin secretion by ribozyme Kir6.2-gene targeting in insulin-secreting cells.

The ATP-sensitive K+ (KATP) channel, composed of Kir6.2 and sulfonylurea receptor (SUR1), in pancreatic beta-cells is believed to serve as a metabolic sensor regulating insulin secretion according to glucose levels. Thus, genetic disruption of Kir6.2 expression may impair KATP channel function in glucose sensing and insulin secretion. Here we show evidence obtained from functional genetic assays supporting this hypothesis. To avoid adaptive cellular mechanisms in transgenic preparations, we designed a hammerhead ribozyme that specifically targeted the Kir6.2 mRNA at serine 78. The Kir6.2-ribozyme was constructed in an adenoviral vector and expressed in insulin-secreting RINm5F cells. Both RT-PCR and Northern blot analyses showed that Kir6.2 transcripts were significantly reduced with a Kir6.2-ribozyme treatment. Whole-cell patch-clamp studies indicated that the Kir6.2-ribozyme treatment lowered KATP channel density by 66%. In response to higher glucose challenge, insulin release from the RINm5F cells dropped by approximately 20% in a transfection dose of 0.7 multiplicity of infection, and by 30-40% in a dose of 2.7 multiplicity of infection. These results therefore indicate that KATP channels play an important role in glucose sensing and insulin secretion, and ribozyme Kir6.2-gene targeting is an effective approach for selective inhibition of functional expression of KATP channels.

Adenoviridae↗

PubMed2004

Molecular basis for the inhibition of G protein-coupled inward rectifier K(+) channels by protein kinase C.

G protein-coupled inward rectifier K(+) (GIRK) channels regulate cellular excitability and neurotransmission. The GIRK channels are activated by a number of inhibitory neurotransmitters through the G protein betagamma subunit (G(betagamma)) after activation of G protein-coupled receptors and inhibited by several excitatory neurotransmitters through activation of phospholipase C. If the inhibition is produced by PKC, there should be PKC phosphorylation sites in GIRK channel proteins. To identify the PKC phosphorylation sites, we performed systematic mutagenesis analysis on GIRK4 and GIRK1 subunits expressed in Xenopus oocytes. Our data showed that the heteromeric GIRK1/GIRK4 channels were inhibited by a PKC activator phorbol 12-myristate 13-acetate (PMA) through reduction of single channel open-state probability. Direct application of the catalytic subunit of PKC to excised patches had a similar inhibitory effect. This inhibition was greatly eliminated by mutation of Ser-185 in GIRK1 and Ser-191 in GIRK4 that remained G protein sensitive. The PKC-dependent phosphorylation seems to mediate the channel inhibition by the excitatory neurotransmitter substance P (SP) as specific PKC inhibitors and mutation of these PKC phosphorylation sites abolished the SP-induced inhibition of GIRK1/GIRK4 channels. Thus, these results indicate that the PKC-dependent phosphorylation underscores the inhibition of GIRK channels by SP, and Ser-185 in GIRK1 and Ser-191 in GIRK4 are the PKC phosphorylation sites.

Animals↗

PubMed2004

Critical protein domains and amino acid residues for gating the KIR6.2 channel by intracellular ATP.

K(ATP) channels couple intermediary metabolism to cellular excitability. Such a property relies on the inherent ATP-sensing mechanism known to be located in the Kir6 subunit. However, the molecular basis for the ATP sensitivity remains unclear. Here we showed evidence for protein domains and amino acid residues essential for the channel gating by intracellular ATP. Chimerical channels were constructed using protein domains of Kir6.2 and Kir1.1, expressed in HEK293 cells, and studied in inside-out patches. The N and C termini, although important, were inadequate for channel gating by intracellular ATP. Full ATP sensitivity also required M1 and M2 helices. Cytosolic portions of the M1 and M2 sequences were crucial, in which six amino acid residues were identified, i.e., Thr76, Met77, Ala161, Iso162, Leu164, and Cys166. Site-specific mutation of any of them reduced the ATP sensitivity. Construction of these residues together with the N/C termini produced ATP sensitivity identical to the wild-type channels. The requirement for specific membrane helices suggests that the Kir6.2 gating by ATP is not shared by even two closest relatives in the K(+) channel family, although the general gating mechanisms involving membrane helices appear to be conserved in all K(+) channels.

Adenosine Triphosphate↗

PubMed2004

Enantiomer separations on a vancomycin stationary phase and retention mechanism of pressurized capillary electrochromatography.

Several chiral drugs, promethazine, carteolol, celiprolol, and albuterol, were resolved with vancomycin as the chiral stationary phase by pressurized capillary electrochromatography (pressurized CEC) and capillary HPLC. The effects of pressure and electrical field strength on efficiency, resolution, and capacity factor in pressurized CEC were investigated. A mathematical model describing the relationship of capacity factor in pressurized CEC with voltage, pressurized flow velocity, electroosmotic mobility, and electrophoretic mobility was established, which was in good agreement with the experimental data.

Chromatography, High Pressure Liquid↗

PubMed2003

Hypercapnic acidosis activates KATP channels in vascular smooth muscles.

ATP-sensitive K+ channels (KATP) couple intermediary metabolism to cellular activity, and may play a role in the autoregulation of vascular tones. Such a regulation requires cellular mechanisms for sensing O2, CO2, and pH. Our recent studies have shown that the pancreatic KATP isoform (Kir6.2/SUR1) is regulated by CO2/pH. To identify the vascular KATP isoform(s) and elucidate its response to hypercapnic acidosis, we performed these studies on vascular smooth myocytes (VSMs). Whole-cell and single-channel currents were studied on VSMs acutely dissociated from mesenteric arteries and HEK293 cells expressing Kir6.1/SUR2B. Hypercapnic acidosis activated an inward rectifier current that was K+-selective and sensitive to levcromakalim and glibenclamide with unitary conductance of approximately 35pS. The maximal activation occurred at pH 6.5 to 6.8, and the current was inhibited at pH 6.2 to 5.9. The cloned Kir6.1/SUR2B channel responded to hypercapnia and intracellular acidification in an almost identical pattern to the VSM current. In situ hybridization histochemistry revealed expression of Kir6.1/SUR2B mRNAs in mesenteric arteries. Hypercapnia produced vasodilation of the isolated and perfused mesenteric arteries. Pharmacological interference of the KATP channels greatly eliminated the hypercapnic vasodilation. These results thus indicate that the Kir6.1/SUR2B channel is a critical player in the regulation of vascular tones during hypercapnic acidosis.

ATP-Binding Cassette Transporters↗

PubMed2003

Orthotopic ileal neobladder similar to original bladder.

OBJECTIVE: To report the surgical techniques and results of an 8-year follow-up study of 42 patients with a modified orthotopic ileal neobladder restoring normal anatomical relationship. METHODS: Total cystoprostatectomy was performed extraperitoneally. A 45 - 50 cm segment of the ileal loop was isolated, detubularized, and reconfigured into an "M"-shape to form a pouch. Bilateral ureters were implanted by inserting 1 cm distal segment into the pouch. The bottom of pouch was opened and anastomosed with the urethra. RESULTS: Forty-two patients were followed up for 6 to 96 months,90.5% of whom were continent in the daytime, and 85.7% at night. Two patients had a difficulty in urination. The average volume of the pouch was (361 +/- 48) ml at 12 months postoperation. Urodynamic examination showed the average peak voiding pressure was (86.8 +/- 21.4) cm H(2)O. The average maximum flow rate (Qmax) was (18.4 +/- 6.1) ml/s. No remarkable ureter reflux and obstruction were found. No patient was detected to have urethral carcinoma. CONCLUSIONS: Extraperitoneal cystectomy can avoid the tumor contamination of the abdomen and intestinal interference of the operative field. The ureter-inserting implantation technique is a simple anti-reflux anastomosis method with less ureter stenosis rate. Isolating the neobladder and ureters from the peritoneal cavity can reduce the postoperative complications, such as adhesive ileus, internal hernia, and urine leakage into the peritoneal cavity. The neobladder is similar to the original bladder in position, volume, shape and anti-reflux ureter connection.

Adult↗

PubMed2002

Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis.

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.

Acidosis↗

PubMed2002

Molecular determinants for activation of G-protein-coupled inward rectifier K+ (GIRK) channels by extracellular acidosis.

Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K(+) (GIRK) channels play a role. To elucidate the effect of extracellular pH (pH(o)) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH(o) to 6.2 augmented GIRK1/GIRK4 currents by approximately 30%. The channel activation was reversible and dependent on pH(o) levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pH(o) 6.2 by approximately 20 and approximately 70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e. H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pH(o) sensitivity. The pH(o) sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%.

Acidosis↗