Carlos G. Onetti

Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in KCNJ8.

BACKGROUND Adenosine triphosphate (ATP)-sensitive potassium cardiac channels consist of inward-rectifying channel subunits Kir6.1 or Kir6.2 (encoded by KCNJ8 or KCNJ11) and the sulfonylurea receptor subunits SUR2A (encoded by ABCC9). OBJECTIVE To examine the association of mutations in KCNJ8 with Brugada syndrome (BrS) and early repolarization syndrome (ERS) and to elucidate the mechanism underlying the gain of function of ATP-sensitive potassium channel current. METHODS Direct sequencing of KCNJ8 and other candidate genes was performed on 204 BrS and ERS probands and family members. Whole-cell and inside-out patch-clamp methods were used to study mutated channels expressed in TSA201 cells. RESULTS The same missense mutation, p.Ser422Leu (c.1265C>T) in KCNJ8, was identified in 3 BrS and 1 ERS probands but was absent in 430 alleles from ethnically matched healthy controls. Additional genetic variants included CACNB2b-D601E. Whole-cell patch-clamp studies showed a 2-fold gain of function of glibenclamide-sensitive ATP-sensitive potassium channel current when KCNJ8-S422L was coexpressed with SUR2A-wild type. Inside-out patch-clamp evaluation yielded a significantly greater half maximal inhibitory concentration for ATP in the mutant channels (785.5 ± 2 vs 38.4 ± 3 μM; n = 5; P <.01), pointing to incomplete closing of the ATP-sensitive potassium channels under normoxic conditions. Patients with a CACNB2b-D601E polymorphism displayed longer QT/corrected QT intervals, likely owing to their effect to induce an increase in L-type calcium channel current (I(Ca-L)). CONCLUSIONS Our results support the hypothesis that KCNJ8 is a susceptibility gene for BrS and ERS and point to S422L as a possible hotspot mutation. Our findings suggest that the S422L-induced gain of function in ATP-sensitive potassium channel current is due to reduced sensitivity to intracellular ATP.

Ischemic preconditioning in the hippocampus of a knockout mouse lacking SUR1-based KATP channels

Background and Purpose— ATP-sensitive K+ (KATP) channels have been implicated in the mechanism of neuronal ischemic preconditioning. To evaluate the role of neuronal/&bgr;–cell-type KATP channels, SUR1 null (Sur1KO) mice lacking (KIR6.x/SUR1)4 KATP channels were subjected to a preconditioning protocol with the use of double carotid occlusion. Methods— Wild-type C57BL/6 and Sur1KO mice were subjected to a double carotid block for 40 minutes with or without a 20-minute preconditioning block. After a 10-day reperfusion period, damage was assessed histologically in the hippocampal CA1, CA2, and CA3 areas and in the dentate gyrus. The neuroprotective effects of intracerebroventricular injections of diazoxide, which selectively affects mitochondria versus opening SUR1-type KATP channels, and 5-hydroxydecanoate, a selective blocker of mitoKATP channels, were evaluated with the same protocol. Results— Neurons in the CA1 region of both Sur1KO and wild-type animals subjected to a 20-minute ischemic insult were protected equally from neuronal damage produced by a subsequent 40-minute ischemic period. Pretreatment with diazoxide protected both Sur1KO and wild-type neurons, while 5-hydroxydecanoate augmented neurodegeneration in both strains of animals when administered before a 20-minute bout of ischemia. Conclusions— SUR1-based KATP channels are not obligatory for neuronal preconditioning or augmentation of neurodegeneration by 5-hydroxydecanoate.

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of γ-aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd2+ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a −50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10−6M [Ca2+]i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from−25 to−10 mV. At 10−9M [Ca2+]i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.

Effects of chronic caffeine administration on blood glucose levels and on glucose tolerance in healthy and diabetic rats.

Objective: To analyse the effect of chronic caffeine use on risk reduction and prognosis of diabetes mellitus. Methods: In this 60-day study, five groups of 11 healthy male Wistar rats were selected to receive one of four doses (37.5, 56.2, 75.0 or 93.0 mg/kg per day) of caffeine orally or no caffeine (control). The effect of caffeine on glycaemia and glucose tolerance was evaluated. After 15 days, each group was treated with 60 mg/kg of streptozotocine to induce diabetes mellitus, and glycaemia and glucose tolerance were assessed for a further 45 days. Results: In nondiabetic rats, caffeine had no effect on blood glucose. Compared with controls, the fasting blood glucose levels declined significantly in two caffeine-treated groups (93.0 mg/kg per day and 56.2 mg/kg per day) during the first 15 days following diabetes induction. Glucose tolerance was significantly improved 120 min after glucose loading in all caffeine-treated groups. The mean ± SE halfmaximal effective concentration of caffeine was 35.79 ± 2.44 mg/dl. Conclusions: Blood glucose levels decreased, and glucose tolerance improved, in diabetic rats administered increasing doses of caffeine.

Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose-sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 × 10-13 cm · s-1. An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physiological K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was — 2 mV. Po decreased noticeably at 50 μM internal adenosine 5′-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 μM. Internal application of 5′-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 μM) was required for the activation of this channel. The glucose—sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.

Biophysical Properties of ATP‐sensitive Potassium Channels in CA3 Hippocampal Neurons

Single‐channel activity of glucose‐sensitive channels from CA3 neurons of the rat hippocampus, was studied in cell‐attached membrane patches. Single‐channel activity was totally abolished at 20 mM external glucose. Glucose‐sensitive channels were selective to K+ ions; the unitary conductance was 170 pS in 140 mM K+, and the K+ permeability was 3.86×10−13 cm⋅s−1. The open‐state probability (PO) increased with membrane depolarization as a result of mean open time enhancement and shortening of the closure periods. The activation midpoint was −79 mV. Glucose‐sensitive K+ channel of CA3 neurons could be considered as an ATP‐sensitive potassium channel.