1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined.Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl-concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10-7.3. The M2 conductance increased as external pH decreased below 8X5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 /SM and 1 /SM, respectively, under physiological conditions.4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-

5. The data were also fitted well by very high values of fl/ca together with a high degree of negative co-operativity or non-equivalence in ACh binding affinity (K2 > K1). A good fit could also be obtained with moderate positive co-operativity combined with non-equivalence of the binding sites.6. A mechanism that postulates a receptor with two independent gating subunits provided a poor fit to the data at negative potential.

Molecular systems that can be remotely controlled by light are gaining increasing importance in cell biology, physiology, and neurosciences because of the spatial and temporal precision that is achievable with laser microscopy. Two-photon excitation has significant advantages deep in biological tissues, but raises problems in the design of "smart" probes compatible with cell physiology. This Review discusses the chemical challenges in generating suitable two-photon probes.

SUMMARY1. Glutamate-receptor ion channels in rat cerebellar granule cells maintained in explant cultures have been investigated with patch-clamp methods. Properties of these channels were determined from noise analysis of whole-cell currents and from noise and single-channel currents recorded in outside-out membrane patches.2. Glutamate (10-20 /LM) evoked two types of response. Some granule cells gave small inward currents accompanied by clear increases in current noise ('large noise' responses), whereas other cells gave larger inward currents and small noise increases ('small noise' responses).3. A mean single-channel conductance (y) of 46-6 pS was estimated for glutamate from four 'large noise' cells. A mean y value of 8-4 pS was estimated for seven other 'large noise' cells. The results suggest that in these latter cells glutamate activated both large (_ 50 pS) and small conductance (-140 fS) channels. 5. Large single-channel currents were evoked by glutamate, aspartate and NMDA in outside-out patches. The mean conductance values obtained for the largest amplitude openings were: y(glutamate) = 49-5 pS, y(aspartate) = 51-5 pS, and y(NMDA) = 53'0 pS. For each agonist, these 50 pS openings comprised 75-85 % of the completely resolved currents in each patch. Openings to 40 and 30pS conductance levels accounted for 10-15 % and 3-7 % of the total, and the presence of apparently direct transitions between these levels and the 50 pS level suggests they are sublevels of the same multi-conductance channels.6. A mean channel conductance of 22-9 pS was estimated from noise evoked by quisqualate (10-30 /SM). Single-channel currents were examined in four patches. In two, quisqualate evoked predominantly small currents of two amplitudes, y = 8&4 pS and 16-5 pS; some 50 pS openings were also present. In the other two patches, most openings were 50 pS events.7. Granule cells gave inward currents to, kainate S. G. CULL-CANDY AND OTHERS channel currents evoked by kainate were of smaller amplitude, with mean conductances of y = 8'1 and 15-1 pS. Kainate also activated 50 pS channels and produced some 30 and 40 pS openings.8. A mean single-channel conductance of 140 fS was estimated from 'small noise' whole-cell currents evoked by glutamate (10-20 /tM). Spectra of noise produced by glutamate in outside-out patches from 'small noise' cells were similar to spectra of noise produced in patches by kainate. 9. Our results indicate that granule cells possess at least three types of glutamatereceptor channels. One type has a maximum conductance of approximately 50 pS;it displays multiple open levels and appears to be activated selectively by aspartate and NMDA. Openings with conductances of 8 and 15 pS are produced by kainate and quisqualate in large numbers and it appears unlikely they are sublevels of the 50 pS channels. Channels with an estimated conductance of 140 fS appear to be present at high density in most granule cells. These channels are activated by glutamate and kainate, but not by aspartate or NMDA to any significant exte...

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−1 (n = 9) at 0.3 μM and 3.66 ± 0.45 WPB s −1 at 100 μM histamine (n = 15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca 2+ indicating that the initial burst of secretion is driven by Ca 2+ release from internal stores, but sustained exocytosis required external Ca 2+ . Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μM), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s (n = 6), and a peak fusion rate of ∼10 WPB s −1 .

The M2 protein of influenza A viruses forms a proton channel involved in modifying virion and trans Golgi pH during infection. Previous studies of the proton current using whole‐cell patch clamp of mouse erythroleukaemia (MEL) cells expressing the M2 protein of the ‘Weybridge’ strain provided evidence for two protonation sites, one involved in permeation, the other in activation by acid pH. The present report compares the M2 channels of two different strains of influenza virus, ‘Weybridge’ (WM2) and ‘Rostock’ (RM2). Whereas with external acid pH the current‐voltage relations showed similar small degrees of inward rectification, a similar apparent Kd of approximately 10 μm for proton permeation and a high selectivity for protons over Na+, the two M2 proteins differed in whole‐cell conductance at low and high pH. The proton conductance of unit membrane area was on average 7‐fold greater in RM2‐ than WM2‐expressing MEL cells. At high external pH WM2 was shown previously to have small conductance for outward current at positive driving potential. In contrast, RM2 shows high conductance for outward current with high external pH, but shows small conductance for inward current with high internal pH, conditions in which WM2 shows high conductance for inward current. The different properties of the conductances due to the two channels at high pH were determined by three amino acids in their transmembrane domains. All intermediate mutants possessed one or other property and transformation of the WM2 phenotype into that of RM2 required substitution in all three residues V27I, F38L and D44N; single substitutions in RM2 effected the opposite phenotypic change. The significance of this difference for virus replication is not clear and it may be that the higher proton flux associated with RM2 is the main factor determining its increased ability to dissipate pH gradients. It is apparent, however, from the specific differences in the sidedness of the pH‐induced changes in voltage dependence of the whole‐cell current that this is an intrinsic property of the virus proton channel which may have parallels with regulation of other proton channels.

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