Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations.

Hexamers of connexins (Cxs) form hemichannels that dock tightly in series via their extracellular domains to give rise to gap junction channels. Here we examined the ability of a variety of C-terminal Cx32 mutations, most of which have been identified in X-linked Charcot-Marie-Tooth disease, to form hemichannels and to complete gap junction channels using the Xenopus oocyte system. First, we show that undocked wild-type Cx32 hemichannels at the plasma membrane can be detected as opening channels activated by depolarization. We have been able to estimate the efficiency of assembly of complete channels by measuring the time-dependent incorporation of preformed hemichannels into gap junction channels after cell-to-cell contact. These data offer strong evidence that hemichannels are the direct precursors of gap junction channels. Of 11 Cx32 mutants tested, a group of 5 mutations prevented the formation of functional hemichannels at the cell surface, whereas 4 mutations were fully able to form precursors but reduced the ability of hemichannels to assemble into complete channels, and 2 mutants formed channels normally. The data revealed that a minimum length of human Cx32 including the residue Arg-215 is required for the expression of hemichannels at the cell surface and that the efficiency of hemichannel incorporation into complete channels decreased gradually with the progressive shortening of the cytoplasmic C-terminal domain.

Molecular basis for different pore properties of potassium channels from the rat brain Kv1 gene family.

Members of the rat brain Kv1 family of cloned potassium channels are structurally highly homologous, but have diverse conductance and pharmacological characteristics. Here we present data on the effects of mutating residues K533 in the P-region and H471 in the S4-S5 linker of Kv1.4 to their equivalent residues in Kv1.1 and Kv1.6 on single-channel conductance and sensitivity to external tetraethylammonium cations (TEA+) and internal Mg2+. Exchange of residue K533 for its equivalent residue (Y) in Kv1.1 and Kv1.6 increased the single-channel conductance at both negative and positive potentials. This mutation is known to reduce the IC50 for external TEA+ from > 100 mM to 0.6 mM, almost identical to that for Kv1.1 (0.53 mM). We have now found that the additional exchange of residue H471 for the equivalent residue (K) in Kv1.6 increased the IC50 for external TEA+ from 0.6 mM (Kv1.4K533Y) to 2.39 mM; this is very close to that for wild-type Kv1.6 channels (2.84 mM). The mutation H471K alone was ineffective. We thus provide evidence that the S4-S5 linker does contribute to the channel's inner-pore region. Data on the block of Kv1 channels by internal Mg2+ indicate that while the binding site is probably situated within the deep-pore region, its exact location may be channel specific.

Calcium dependence and distribution of calcium-activated chloride channels in Xenopus oocytes.

1. The Ca(2+)-dependent Cl- current (ICl,Ca), expressed in the plasma membrane of Xenopus oocytes, was examined in excised inside-out macropatches using a rapid perfusion system. 2. Application of Ca(2+)-containing Ringer solution resulted in the activation of a current whose reversal potential shifted to the right by 51 +/- 5.2 mV when Cl- in the pipette solution was lowered from 119.3 to 10 mM. No currents were generated when Ca2+ was omitted from the solution. The current is therefore a Ca(2+)-activated Cl- one. 3. Following exposure to Ca2+, the half-time for activation of ICl,Ca was not voltage dependent, whereas deactivation was strongly so. 4. ICl,Ca was stable in the continuous presence of Ca2+ and showed no sign of inactivation or adaptation. 5. Comparison of the size of the currents (normalized to pipette resistance) from the animal and vegetal poles revealed that ICl,Ca had a highly polarized distribution. The current density was almost 10 times higher in the animal pole. 6. The results suggest that Cl- channels provide a continuous and reliable indication of submembranous Ca2+, at least in an excised patch, and the clustering of the Cl- channels renders it necessary to exert caution in interpreting results involving the kinetics of Ca2+ signalling, when ICl,Ca is used as the sole monitor of calcium.

Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue.

We isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X4 receptor (87% identity). By fluorescence in situ hybridization, the human P2X4 gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X4 transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X4 receptor in Xenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embronic kidney 293 cells transfected with human P2X4 cDNA allowed us to determine the fraction of the current carried by Ca2: this was approximately 8%, demonstrating a high Ca2+ permeability. Low extracellular Zn2+ concentrations (5-10 microM) increase the apparent gating efficiency of human P2X4 by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (> 100 microM) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X4 receptor displays a very similar agonist potency profile to that of rat P2X4 (ATP > > 2-methylthio-ATP > or = CTP > alpha, beta-methylene-ATP > dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid.

P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues.