The IsoStretcher: An isotropic cell stretch device to study mechanical biosensor pathways in living cells.

Mechanosensation in many organs (e.g. lungs, heart, gut) is mediated by biosensors (like mechanosensitive ion channels), which convert mechanical stimuli into electrical and/or biochemical signals. To study those pathways, technical devices are needed that apply strain profiles to cells, and ideally allow simultaneous live-cell microscopy analysis. Strain profiles in organs can be complex and multiaxial, e.g. in hollow organs. Most devices in mechanobiology apply longitudinal uniaxial stretch to adhered cells using elastomeric membranes to study mechanical biosensors. Recent approaches in biomedical engineering have employed intelligent systems to apply biaxial or multiaxial stretch to cells. Here, we present an isotropic cell stretch system (IsoStretcher) that overcomes some previous limitations. Our system uses a rotational swivel mechanism that translates into a radial displacement of hooks attached to small circular silicone membranes. Isotropicity and focus stability are demonstrated with fluorescent beads, and transmission efficiency of elastomer membrane stretch to cellular area change in HeLa/HEK cells. Applying our system to lamin-A overexpressing fibrosarcoma cells, we found a markedly reduced stretch of cell area, indicative of a stiffer cytoskeleton. We also investigated stretch-activated Ca(2+) entry into atrial HL-1 myocytes. 10% isotropic stretch induced robust oscillating increases in intracellular Fluo-4 Ca(2+) fluorescence. Store-operated Ca(2+) entry was not detected in these cells. The Isostretcher provides a useful versatile tool for mechanobiology.

Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Ca(v)beta subunits.

N-type and P/Q-type Ca(2+) channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gbetagamma subunits. Inhibition is caused by a shift from an easily activated "willing" (W) state to a more-difficult-to-activate "reluctant" (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca(2+) channels containing Cav2.2a alpha(1) subunits and P/Q-type Ca(2+) channels containing Ca(v)2.1 alpha(1) subunits revealed substantial differences. In the absence of G protein modulation, Ca(v)2.1 channels containing Ca(v)beta subunits were tonically in the W state, whereas Ca(v)2.1 channels without beta subunits and Ca(v)2.2a channels with beta subunits were tonically in the R state. Both Ca(v)2.1 and Ca(v)2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca(v)beta subunits are intrinsic properties of the Ca(2+) channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca(2+)-channel activity incorporating an intrinsic equilibrium between the W and R states of the alpha(1) subunits and modulation of that equilibrium by G proteins, Ca(v)beta subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca(2+) channels to initiate neurotransmitter release.

G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins.

1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.

G-protein mediated gating of inward-rectifier K+ channels.

G-protein regulated inward-rectifier potassium channels (GIRK) are part of a superfamily of inward-rectifier K+ channels which includes seven family members. To date four GIRK subunits, designated GIRK1-4 (also designated Kir3.1-4), have been identified in mammals, and GIRK5 has been found in Xenopus oocytes. GIRK channels exist in vivo both as homotetramers and heterotetramers. In contrast to the other mammalian GIRK family members, GIRK1 can not form functional channels by itself and has to assemble with GIRK2, 3 or 4. As the name implies, GIRK channels are modulated by G-proteins; they are also modulated by phosphatidylinositol 4,5-bisphosphate, intracellular sodium, ethanol and mechanical stretch. Recently a family of GTPase activating proteins known as regulators of G-protein signaling were shown to be the missing link for the fast deactivation kinetics of GIRK channels in native cells, which contrast with the slow kinetics observed in heterologously expressed channels. GIRK1, 2 and 3 are highly abundant in brain, while GIRK4 has limited distribution. Here, GIRK1/2 seems to be the predominant heterotetramer. In general, neuronal GIRK channels are involved in the regulation of the excitability of neurons and may contribute to the resting potential. Interestingly, only the GIRK1 and 4 subunits are distributed in the atrial and sinoatrial node cells of the heart and are involved in the regulation of cardiac rate. Our main objective of this review is to assess the current understanding of the G-protein modulation of GIRK channels and their physiological importance in mammals.

Regulation of GIRK channel deactivation by Galpha(q) and Galpha(i/o) pathways.

G protein regulated inward rectifying potassium channels (GIRKs) are activated by G protein coupled receptors (GPCRs) via the G protein betagamma subunits. However, little is known about the effects of different GPCRs on the deactivation kinetics of transmitter-mediated GIRK currents. In the present study we investigated the influence of different GPCRs in the presence and absence of RGS proteins on the deactivation kinetics of GIRK channels by coexpressing the recombinant protein subunits in Xenopus oocytes. The stimulation of both G(i/o)- and G(q)-coupled pathways accelerated GIRK deactivation. GIRK currents deactivated faster upon stimulation of G(i/o)- and G(q)-coupled pathways by P(2)Y(2) receptors (P(2)Y(2)Rs) than upon activation of the G(i/o)-coupled pathway alone via muscarinic acetylcholine receptor M2 (M(2) mAChRs). This acceleration was found to be dependent on phospholipase C (PLC) and protein kinase C (PKC) activities and intracellular calcium. With the assumption that RGS2 has a higher affinity for Galpha(q) than Galpha(i/o), we demonstrated that the deactivation kinetics of GIRK channels can be differentially regulated by the relative amount of RGS proteins. These data indicate that transmitter-mediated deactivation of GIRK currents is modulated by crosstalk between G(i/o)- and G(q)-coupled pathways.

Synaptic localization and presynaptic function of calcium channel beta 4-subunits in cultured hippocampal neurons.

Neurotransmitter release is triggered by the influx of Ca(2+) into the presynaptic terminal through voltage gated Ca(2+)-channels. The shape of the presynaptic Ca(2+) signal largely determines the amount of released quanta and thus the size of the synaptic response. Ca(2+)-channel function is modulated in particular by the auxiliary beta-subunits that interact intracellularly with the pore-forming alpha(1)-subunit. Using retrovirus-mediated gene transfer in cultured hippocampal neurons, we demonstrate that functional GFP-beta(4) constructs colocalize with the synaptic vesicle marker synaptobrevin II and endogenous P/Q-type channels, indicating that beta(4)-subunits are localized to synaptic sites. Costaining with the dendritic marker MAP2 revealed that the beta(4)-subunit is transported to dendrites as well as axons. The nonconserved amino- and carboxyl-termini of the beta(4)-subunit were found to target the protein to the synapse. Physiological measurements in autaptic hippocampal neurons infected with green fluorescent protein (GFP)-beta(4) revealed an increase in both excitatory post-synaptic current amplitude and paired pulse facilitation ratio, whereas the GFP-beta(4) mutant, GFP-beta(4)(Delta50-407), which demonstrated a cytosolic localization pattern, did not alter these synaptic properties. In summary, our data suggest a pre-synaptic function of the Ca(2+)-channel beta(4)-subunit in synaptic transmission.

New roles for RGS2, 5 and 8 on the ratio-dependent modulation of recombinant GIRK channels expressed in Xenopus oocytes.

1. The activation of G protein-regulated inward rectifying potassium (GIRK) channels is modulated by G protein-coupled receptors (GPCRs) via the G protein betagamma subunits and is accelerated by regulators of G protein signalling (RGS). In the present study we investigated the ratio dependence of receptor-mediated activation and deactivation and the influence of new members of the RGS protein family on GIRK currents by coexpressing the recombinant protein subunits in Xenopus oocytes and further analysis of the whole cell currents. 2. The activation of GIRK channels by the muscarinic acetylcholine receptor M2 (M2 mAChR) is strongly dependent on the ratio of receptor to channel in Xenopus oocytes. The increase and on-rate of the amplified current is affected by this ratio. An excess of receptor over channel is necessary for current amplification, while the reverse excess of channel over receptor abolishes the effect. 3. The speed of receptor-mediated activation of GIRK currents is accelerated for a high ratio of receptor to channel, while the time of deactivation is independent of this ratio. 4. Coexpression of RGS2, 5 and 8 accelerates the speed for ACh-mediated activation and deactivation of GIRK1/2 and GIRK1/4 currents. Thereby the receptor/channel/RGS ratio determines the amount of current amplification. 5. Bordetella pertussis toxin completely abolished ACh-mediated current amplification of GIRK channels coexpressed with or without RGS2. 6. Two single point mutations in the RGS2 protein (RGS2(N109S) and RGS2(L180F)) reduced the acceleration of current amplification after ACh application on GIRK1/4 channels compared with RGS2 wild-type protein.

PIP2 and PIP as determinants for ATP inhibition of KATP channels.

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel alpha1A subunit.

Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein betagamma subunits that modulate presynaptic Ca2+ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein betagamma subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L I-II) of Ca2+ channel alpha1A subunits, which are modulated by G proteins, but not in alpha1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from alpha1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from alpha1A can competitively inhibit Gbetagamma modulation. Conversion of the R in the QQIER sequence of alpha1A to E as in alpha1C slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials. Conversion of the final E in the QQLEE sequence of alpha1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for alpha1A. Mutation of the QQIER sequence in alpha1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in alpha1C greatly reduced G protein modulation and increased the rate of reversal of G protein effects. These results indicate that the QXXER motif in L I-II is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large influence on modulation by Gbetagamma. Overlap of this motif with the consensus sequence for binding of Ca2+ channel beta subunits suggests that this region of L I-II is important for three different modulatory influences on Ca2+ channel activity.

Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors.

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the gamma-subunit of the fetal muscle acetylcholine receptor (gamma-AChR) is replaced by the epsilon-subunit in the adult muscle receptor (epsilon-AChR), was investigated by analysing the structure of gamma- and epsilon-subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single-channel conductance. 2. The gamma- and epsilon-subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilon(gamma)- or gamma(epsilon)-subunits were constructed from the parental epsilon- and gamma-subunits, respectively. Exchanging the four hydrophobic segments (M1-M4) of the gamma-subunit for those of the epsilon-subunit and vice versa completely reversed the difference in conductance between gamma-AChR and epsilon-AChR channels. 4. Effects of single- and multiple-point mutations in M1-M4 segments of gamma- and epsilon-subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (gamma-subunit) for serine/isoleucine (epsilon-subunit) in M2, and the lysine (gamma-subunit) for glutamine (epsilon-subunit) exchanges in the regions flanking the M2 segment.

Modulation of Ca2+ channels by G-protein beta gamma subunits.

Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.

Short antisense oligonucleotide-mediated inhibition is strongly dependent on oligo length and concentration but almost independent of location of the target sequence.

The inhibitory effect of short antisense oligodeoxynucleotides (aODNs) on cRNA expression in Xenopus oocytes was measured using an electrophysiological assay based on subunit-specific block of cloned alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. The effect of both phosphorothioate-modified (PS) and phosphodiester (PO) aODNs was strongly length dependent with a half-maximal inhibition calculated for an oligo length of 7.6 nucleotides (nt) and 9.9 nt, respectively. More than 95% inhibition was mediated by a PS aODN of 12 nt and by PO aODNs > or = 15 nt. At a given length PS and PO aODNs showed differential dependence of their inhibitory effect on the injected aODN concentration (half-maximal inhibition at 18 ng/microliter for a PO 12-mer and at 0.19 ng/microliter for a PS 12-mer) and differential saturation behavior. The inhibitory effect of aODNs, even as short as 8 nt for PS oligomers, was highly sequence specific, but almost independent of the position of the respective target site on the cRNA (for PS 8-mers, > or = 70% expression inhibition throughout the tested target sites from the translation initiation to the 3'-untranslated region). Thus, short PS aODNs can be reliably used in order to specifically inhibit protein expression in experiments addressing physiological, molecular biological, and perhaps even therapeutical issues.

Argiotoxin detects molecular differences in AMPA receptor channels.

Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AMPA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.

Location of a threonine residue in the alpha-subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel.

By the combination of cDNA manipulation and functional analysis of normal and mutant acetylcholine receptor (AChR) channels of Torpedo expressed in Xenopus laevis oocytes determinants of ion flow were localized in the bends bordering the putative M2 transmembrane segment (Imoto et al. 1988). We now report that in the rat muscle AChR, substitution of a threonine residue in the alpha-subunit localized in the M2 transmembrane segment increases or decreases the channel conductance, depending on the size of the amino acid side chain located at this position. This threonine residue (alpha T264) is located adjacent to the cluster of charged amino acids that form the intermediate anionic ring (Imoto et al. 1988). This effect is pronounced for the large alkali cations Cs+, Rb+, K+ whereas for Na+ the effect is much smaller. Taken together the results suggest that the threonine residues at position 264 in the two alpha-subunits together with the amino acids of the intermediate anionic ring form part of a narrow region close to the cytoplasmic mouth of the AChR channel.

A general and rapid mutagenesis method using polymerase chain reaction.

The construction of deletions, insertions and point mutations in DNA sequences is a powerful approach to analysing the function and structure of genes and their products. Here, we present a fast and efficient method using the polymerase chain reaction to introduce mutations into cDNAs coding for the alpha-, gamma- and epsilon-subunit of the rat muscle acetylcholine receptor. Two flanking primers and one mutant oligo, in conjunction with supercoiled plasmid DNA and a fragment of the target DNA are sufficient to introduce the mutation by two PCR amplifications. Our method permits directing the location of mutations anywhere in the target gene with a very low misincorporation rate, as no substitution could be detected within 9600 bp. The utility of this approach is demonstrated by the rapid introduction and analysis of eleven mutations into three different cDNAs. Any kind of mutation can be introduced with an efficiency of at least 50%.