Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 ± 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Characterization of a high-voltage-activated IA current with a role in spike timing and locomotor pattern generation

Transient A-type K+ channels (IA) in neurons have been implicated in the delay of the spike onset and the decrease in the firing frequency. Here we have characterized biophysically and pharmacologically an IA current in lamprey locomotor network neurons that is activated by suprathreshold depolarization and is specifically blocked by catechol at 100 μM. The biophysical properties of this current are similar to the mammalian Kv3.4 channel. The role of the IA current both in single neuron firing and in locomotor pattern generation was analyzed. The IA current facilitates Na+ channel recovery from inactivation and thus sustains repetitive firing. The role of the IA current in motor pattern generation was examined by applying catechol during fictive locomotion induced by N-methyl-d-aspartate. Blockade of this current increased the locomotor burst frequency and decreased the firing of motoneurons. Although an alternating motor pattern could still be generated, the cycle duration was less regular, with ventral roots bursts failing on some cycles. Our results thus provide insights into the contribution of a high-voltage-activated IA current to the regulation of firing properties and motor coordination in the lamprey spinal cord.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-ΔF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the βgal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.

Depletion of intracellular polyamines relieves inward rectification of potassium channels.

Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches.

Probing the pore region of recombinant N-methyl-D-aspartate channels using external and internal magnesium block.

Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations of residues five to seven positions downstream of the Q/R/N site do not influence the external Mg2+ block, but clearly influence the block by internal Mg2+. These data add support to the hypothesis that there are two separate binding sites for external and internal Mg2+ block. They also indicate that the C-terminal end of TMII contributes to the inner vestibule of the pore of NMDA channels and thus provide additional evidence that TMII forms a loop that reemerges toward the cytoplasmic side of the membrane.

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

Voltage gating and permeation in a gap junction hemichannel.

Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained from Xenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, gammaopen, to a substate, gammasub, about one-third of the conductance of gammaopen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, gammaclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between gammaclosed and gammaopen appear slow (10-20 ms), often involving several transient substates distinct from gammasub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (approximately 8.5 angstroms diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.