N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two new crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1(L)) and the G protein-sensitive Kir3.1 (Kir3.1(S)) channels in the absence of PIP(2) show the cytoplasmic ion-permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1(L) and Kir3.1(S) suggests a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structural comparison shows a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1(L) that is important for modulating inward rectification. Taken together, these results suggest the cytoplasmic domains of Kir channels undergo structural changes to modulate gating and inward rectification.

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.

A B ST RAC TSodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Ql0 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (/Ca) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 IxM to < 0.3 ~M. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (el) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 JiM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (~ = 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased.

Dynamic responses of cardiac sodium-calcium exchange current to changes of cytoplasmic calcium and MgATP were monitored and analyzed in giant membrane patches excised from guinea pig myocytes. Secondary dependencies of exchange current on cytoplasmic calcium are accounted for in terms of two mechanisms: (a) The sodium-dependent inactivation process, termed Ii modulation, is itself strongly modulated by cytoplasmic calcium. Recovery from the I1 inactivated state is accelerated by increasing cytoplasmic calcium, and the calculated rate of entrance into I1 inactivation is slowed. (b) A second modulation process, termed I.o modulation, is not sodium dependent. As with Il modulation, the entrance into I2 inactivation takes place over seconds in the absence of cytoplasmic calcium. The recovery from I2 inactivation is a calcium-dependent transition and is rapid (< 200 ms) in the presence of micromolar free calcium. Ii and I2 modulation can be treated as linear, independent processes to account for most exchange modulation patterns observed: (a) When cytoplasmic calcium is increased or decreased in the presence of high cytoplasmic sodium, outward exchange current turns on or off, respectively, on a time scale of multiple seconds. (b) When sodium is applied in the absence of cytoplasmic calcium, no outward current is activated. However, the flail outward current is activated within solution switch time when cytoplasmic calcium is applied together with sodium. (c) The calcium dependence of peak outward current attained upon application of cytoplasmic sodium is shifted by ~ 1 log unit to lower concentrations from the calcium dependence of steady-state exchange current. (d) The time course of outward current decay upon decreasing cytoplasmic calcium becomes more rapid as calcium is reduced into the submicromolar range. (e) Under nearly all conditions, the time courses of current decay during application of cytoplasmic sodium and/or removal of cytoplasmic calcium are well fit by single exponentials. Both of the modulation processes are evidently affected by MgATP.Address reprint requests to Donald W. Hilgemann,

SUMMARY1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na+-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 GQ seals with 14-24 jum pipette tip diameters) excised from guineapig, rabbit and mouse myocytes.2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads.3. To further establish the validity of the method for ion transport studies, measurements of Na+-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 Ium), (iii) fast, rigorous concentration control and (iv) Na+-K+ pump densities in the range of whole-cell densities.4. Stimulation of outward Na+-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current.5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (halftime) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 gM-free calcium).6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased.7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mm or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mm or greater. MS 8777A. COLLINS, A. V. SOMLYO AND D. W. HILGEMANN 8. Stimulation of Na'-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mm-EGTA.9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 uM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.10. The stimulatory effect of MgATP was not mimicked by MgATP-y-S, and it was not reversed by acid phosphatase, alkaline phosphatase or an isolated cardiac protein phosphatase. Further, the effect was not enhanced nor was decay of the effect prolonged by 2 /LM of the phosphatase inhibitor, okadaic acid.11. We conclude that stimulation of Na'-Ca2' exchange current in excised sarcolemmal patches by MgATP is not a calcium-dependent process and probably does not involve protein kin...

Spinal and hip osteopenia and vertebral fractures are a feature of mild AS. However, there was no correlation between BMD and vertebral fractures in these patients. AS patients with mild disease had a higher risk of fractures compared with the normal population and this increased with the duration of disease.

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Thermography can be used for the assessment of joint involvement in inflammatory arthritis. Modem thermographic apparatus allows temperature to be measured to within 0-2°C. and simultaneously records the areas involved. It has been demonstrated that thermographic changes in skin temperature over areas of chronic inflammation properly reflect changes in other chemical and cellular processes in rheumatoid arthritis (Ring and Collins, 1970;Collins and Cosh, 1970). The method also agrees with the findings of joint scans after technetium injection (Cosh, Lindsay, Rhys-Davies, and Ring, 1970) and correlates with the intra-articular temperature taken by thermistor probe (Lloyd-Williams, Ring, and Cosh, 1970). Recently Pinder and Ring (1974) have shown that thermographic recordings accurately locate areas of inflamed synovial tissue as shown at synovectomy.The effect of anti-inflammatory compounds on joint inflammation in animals has been quantitated using radiometry (Collins and Ring, 1972). Thermography provides more information about the distribution of temperature. We wish to report a method for the quantitative measurement of joint inflammation in man, using thermography. Method THERMOGRAPHIC APPARATUSThe apparatus used was the Bofors Thermograph (Ring, 1971). The thermographic image was photographed from the cathode ray display tube on to 35 mm. colour film. Isothermal patterns, at 0 5°C. intervals, were photographed through coloured filters to produce a single multiisothermal picture of skin temperature. Each isothermal area was represented by a separate colour. All thermograms were taken in a special room which was draught-free, shielded from direct sunlight, and maintained at a constant temperature of 20°C. The patients were seated in this controlled environment, with the limb to be examined unclothed, for not less than 20 minutes before thermography. Patients having undergone recent physiotherapy were excluded. On each occasion, themagnification ofthe image