Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

High-resolution structure of hair-cell tip links

Transduction-channel gating by hair cells apparently requires a gating spring, an elastic element that transmits force to the channels. To determine whether the gating spring is the tip link, a filament interconnecting two stereocilia along the axis of mechanical sensitivity, we examined the tip link's structure at high resolution by using rapid-freeze, deep-etch electron microscopy. We found that the tip link is a right-handed, coiled double filament that usually forks into two branches before contacting a taller stereocilium; at the other end, several short filaments extend to the tip link from the shorter stereocilium. The structure of the tip link suggests that it is either a helical polymer or a braided pair of filamentous macromolecules and is thus likely to be relatively stiff and inextensible. Such behavior is incompatible with the measured elasticity of the gating spring, suggesting that the gating spring instead lies in series with the helical segment of the tip link.

A model for amplification of hair-bundle motion by cyclical binding of Ca2+ to mechanoelectrical-transduction channels

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca2+ to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system’s behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca2+ binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken’s cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a “Deg” mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

Putting ion channels to work: Mechanoelectrical transduction, adaptation, and amplification by hair cells

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer's adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.

Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.

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.

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.

Activity of primate inferotemporal neurons related to a sought target in pair-association task.

Visual long-term memory in primates has been assessed by using the pair-association (PA) task, in which a subject retrieves and chooses the paired associate of a cue picture. Our previous studies on single neurons in the anterior inferotemporal (AIT) cortex suggested their roles in representing paired associates in the mind. To test the possibility that the delay activity of AIT neurons is related to a particular picture as a sought target, we devised the PA with color switch (PACS) task. In the PACS task, the necessity for memory retrieval and its initiation time were controlled by a color switch in the middle of the delay period. A control task, in which there is no color switch, corresponds to the conventional delayed matching-to-sample (DMS) task where the monkey chooses the same picture as a cue. We found that AIT neurons started to respond just after the color switch in the PACS task, when the cue-optimal picture's associate was presented as a cue. In contrast, they showed no response change in the DMS task. We confirmed that this effect is not due to the visual response to colors. Furthermore, when the cue-optimal picture was presented as a cue, these neurons showed suppression after the color switch in the PACS task. These results suggest that the activity of AIT neurons mediates gating mechanisms that preferentially pass information about a sought target, even when the sought target is retrieved from long-term memory.

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.

Zn2+ interaction with Alzheimer amyloid beta protein calcium channels.

The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.

Serine-1321-independent regulation of the mu 1 adult skeletal muscle Na+ channel by protein kinase C.

The adult skeletal muscle Na+ channel mu1 possesses a highly conserved segment between subunit domains III and IV containing a consensus protein kinase C (PKC) phosphorylation site that, in the neuronal isoform, acts as a master control for "convergent" regulation by PKC and cAMP-dependent protein kinase. It lacks an approximately 200-aa segment between domains I and II though to modulate channel gating. We here demonstrate that mu1 is regulated by PKC (but not cAMP-dependent protein kinase) in a manner distinct from that observed for the neuronal isoforms, suggesting that under the same conditions muscle excitation could be uncoupled from motor neuron input. Maximal phosphorylation by PKC, in the presence of phosphatase inhibitors, reduced peak Na+ currents by approximately 90% by decreasing the maximal conductance, caused a -15 mV shift in the midpoint of steady-state inactivation, and caused a slight speeding of inactivation. Surprisingly, these effects were not affected by mutation of the conserved serine (serine-1321) in the interdomain III-IV loop. the pattern of current suppression and gating modification by PKC resembles the response of muscle Na+ channels to inhibitory factors present in the serum and cerebrospinal fluid of patients with Guillain-Barré syndrome, multiple sclerosis, and idiopathic demyelinating polyradiculoneuritis.

Subunit interactions in coordination of Ni2+ in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels present a unique model for studying the molecular mechanisms of channel gating. We have studied the mechanism of potentiation of expressed rod CNG channels by Ni2+ as a first step toward understanding the channel gating process. Here we report that coordination of Ni2+ between histidine residues (H420) on adjacent channel subunits occurs when the channels are open. Mutation of H420 to lysine completely eliminated the potentiation by Ni2+ but did not markedly alter the apparent cGMP affinity of the channel, indicating that the introduction of positive charge at the Ni(2+)-binding site was not sufficient to produce potentiation. Deletion or mutation of most of the other histidines present in the channel did not diminish potentiation by Ni2+. We studied the role of subunit interactions in Ni2+ potentiation by generating heteromultimeric channels using tandem dimers of the rod CNG channel sequence. Injection of single heterodimers in which one subunit contained H420 and the other did not (wt/H420Q or H420Q/wt) resulted in channels that were not potentiated by Ni2+. However, coinjection of both heterodimers into Xenopus oocytes resulted in channels that exhibited potentiation. The H420 residues probably occurred predominantly in nonadjacent subunits when each heterodimer was injected individually, but, when the two heterodimers were coinjected, the H420 residues could occur in adjacent subunits as well. These results suggest that the mechanism of Ni2+ potentiation involves intersubunit coordination of Ni2+ by H420. Based on the preferential binding of Ni2+ to open channels, we suggest that alignment of H420 residues of neighboring subunits into the Ni(2+)-coordinating position may be associated with channel opening.