pICln inhibits snRNP biogenesis by binding core spliceosomal proteins.

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.

Evidence for direct physical association between a K+ channel (Kir6.2) and an ATP-binding cassette protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K+ channel.

Structurally unique among ion channels, ATP-sensitive K+ (KATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K+ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2deltaC37). Kir6.2deltaC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2deltaC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 exhibited single-channel activity characteristic of pancreatic KATP channels. Kir6.2deltaC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K+ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a KATP channel.

Molecular mechanisms of photoreception. IV. Ca2+-inhibited GTPase of rod outer segments of the frog retina.

Ca2+-dependent GTPase activity is found to be present in the rod outer segments of frog retina. GTPase localization in rod outer segments is shown by fractionating the rod outer segment preparation in the sucrose density gradient. The enzyme is readily washed out of cells with isotonic NaCl solution. The Km is 0.6 mM for GTP. The activity is inhibited by 78 +/- 12% with the increase in Ca2+ concentration from 10(-9) to 10(-7) M. GTP hydrolysis is inhibited by the same concentrations of Ca2+ which block the sodium conductivity of the rod outer segment cytoplasmic membrane.

Antiidiotypic antibodies against anti-cGMP polyclonal antibodies.

Affinity-purified polyclonal anti-cGMP antibodies were obtained from rabbit serum after immunization by succinyl derivative of cGMP coupled to bovine serum albumin. These antibodies were used to raise antiidiotypic antibodies in rats. Putative antiidiotypic serum inhibited the binding of [3H]cGMP to affinity-purified anti-cGMP antibodies. The influence of immunoglobulins isolated from antiidiotypic serum on the ion conductance of rod outer segment plasma membrane fragments from frog retina was studied in patch-clamp experiments. These immunoglobulins increased the conductance of ion channels acting like a natural agonist (cGMP). Preimmune immunoglobulins did not act. The data obtained suggest that antiidiotypic antibodies interact with regulatory cGMP-binding sites of the plasma membrane channels.

Low molecular mass phosphoproteins from the frog rod outer segments form a complex with 48 kDa protein.

Upon separation of cAMP-dependent low molecular mass phosphoproteins [Components I and II; Polans et al. (1979) J. gen. Physiol. 74, 595-613] from the frog rod outer segments by gel-chromatography, isoelectric focusing, non-denaturating electrophoresis and ion-exchange chromatography, they behave like subunits of the oligomeric complex. Apparent molecular mass of the complex determined by gel-chromatography is 52-57 kDa and by non-denaturating gradient electrophoresis is 62-66 kDa. The isoelectric point of the complex is 5.5. The elution profile of Components I and II upon gel-chromatography and ion-exchange chromatography coincides with that of major rod outer segment 48 kDa protein. The isoelectric point for them also coincides with the isoelectric point of 48 kDa protein. The amount of low molecular mass phosphoproteins is sealed rods is equal to one molecule per 60 rhodopsin molecules and coincides with that of a 48 kDa protein. It is suggested that in solution Components I and II form an oligomeric complex with 48 kDa protein.

GTP-binding protein associated with amino acid binding proteins from olfactory epithelium of skate, Dasyatis pastinaca.

Amino acid binding protein (98 kDa) from olfactory epithelium of skate, Dasyatis pastinaca, form a stable complex with the 56 kDa protein. This complex dissociates in the presence of 10 microM GTP gamma S and 2 mM MgCl2. The 56 kDa protein has a steady-state GTPase activity (15 nmol/min per mg). Binding of amino acids to the 98 kDa protein specifically stimulates GTPase activity of the 56 kDa protein; half-maximal stimulation of GTPase activity is observed at 0.1 microM amino acid.

Regulation of cGMP-dependent conductance in cytoplasmic membrane of rod outer segments by transducin.

A preparation of the photoreceptor G-protein, transducin, containing mainly the T alpha-subunit in a GTP-gamma-S-bound form, has been used for perfusion of the intracellular surface of excised patches of rod outer segment cytoplasmic membrane from frog retina. The preparation has been shown to result in the complete suppression of the cGMP-activated ionic conductance of the cytoplasmic membrane patch. The effect is entirely reversible after the protein has been washed out and is not observed in the absence of cGMP. The degree of conductance inhibition depends on the protein concentration, half-maximal inhibition occurring at 1 microM T alpha-GTP-gamma-S.

Immobilization of immunoglobulin on a quartz surface by BrCN without loss of antigen binding capability.

BrCN-activated quartz was used for the immobilization of monoclonal immunoglobulins. BrCN-activated quartz keeps its ability to quantitatively bind immunoglobulins for at least 8 h. The surface density of the immobilized immunoglobulins was 100 ng cm-2 and they were found to retain their ability to bind antigen in a molar ratio of 1:2 for not less than three months.

Characterization of a native swelling-induced chloride current, ICl.swell, and its regulatory protein, pICln, in Xenopus oocytes.

The ability to precisely regulate cell volume is a fundamental property of most cells. Although the phenomenon of regulatory volume decrease (RVD), whereby a swollen cell loses salt and water to restore its original volume, has been appreciated for decades, the molecular identities of the proteins responsible for the volume control machinery and their regulation are essentially unknown. It appears that the rate-determining step in gaining volume control involves the activation of potassium and chloride conductance pathways. We have identified a native chloride current (ICl.swell) responsive to cell swelling in Xenopus oocytes [Ackerman et al. (1994) J Gen Physiol 103: 153-179]. Moreover, we have demonstrated that a cloned protein, pICln, endogenous to oocytes is critical for the activation of this volume-sensitive chloride conductance pathway [Krapivinsky et al. (1994) Cell 76: 439-448]. The identification of an endogenous protein participating in the regulation of an endogenous current may help understand the physiological activities of swelling-induced chloride channels.

Antiidiotypic antibodies interacting with cGMP-dependent channels of frog retinal rod outer segments.

Antiidiotypic approach was used to obtain antibodies interacting with cGMP-binding site of the cGMP-activated channel of the photoreceptor cell. Monoclonal anti-BrcGMP antibodies having characteristics of binding of agonist and its analogs close to those for a natural receptor have been obtained. These antibodies were used to raise polyclonal antiidiotypic antibodies capable of interacting with a natural cGMP-receptor. Application of immunoglobulins, isolated from antiidiotypic serum, to inside-out fragments of the rod plasma membrane led to an irreversible increase of the conductance of cGMP-dependent channels.

Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICln.

Cells maintain control of their volume by the passage of KCl and water across their membranes, but the regulatory proteins are unknown. Expression in Xenopus oocytes of a novel protein, pICln, activated a chloride conductance. We have cloned analogs of pICln from rat heart and Xenopus ovary. pICln was identified as an abundant soluble cytosolic protein (approximately 40 kd) that does not immunolocalize with the plasma membrane. pICln was found in epithelial and cardiac cells, brain, and Xenopus oocytes, forming complexes with soluble actin and other cytosolic proteins. Monoclonal antibodies recognizing pICln blocked activation of a native hypotonicity-induced chloride conductance (ICl.swell) in Xenopus oocytes, suggesting that pICln may link actin-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. The high degree of sequence conservation and widespread expression of pICln suggest that it is an important element in cellular volume regulation.

Recombinant G-protein beta gamma-subunits activate the muscarinic-gated atrial potassium channel.

Acetylcholine activates inwardly rectifying potassium channels (IK.ACh) in the heart through muscarinic receptor binding and activation of pertussis-toxin-sensitive G proteins. Experiments showing that only the beta gamma-subunit (G beta gamma) activates IK.ACh (ref. 4) were challenged by reports that only the activated alpha-subunit (G alpha) was effective. Here we examine IK.ACh regulation using purified brain and recombinant G-protein subunits. Six recombinant G beta gamma-subunits activated IK.ACh with apparent half-maximal activation concentrations of 3-30 nM. Activation of IK.ACh by recombinant G alpha-GTP gamma S was observed, but this was probably due to release of GTP gamma S from the protein. Importantly, IK.ACh activity elicited by GTP gamma S was inhibited by purified brain and recombinant G alpha-GDP, suggesting that native G beta gamma plays a major role in this pathway. We conclude that G beta gamma is a primary regulator of IK.ACh activity.