Biomedical subjects
Publications and source records attributed to S V Ramanan.
The Adventures of Sherlock Holmes by Sir Arthur Conan Doyle contains many incidents of medical interest. While disorders of the cardiovascular system do not play an important role in these tales, there are, nevertheless, some illnesses that invite speculation. Eleven such incidents are reviewed and discussed in light of the times in which they occurred and in light of current medical knowledge.
Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254Delta293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254Delta293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics.
The single-channel conductance of the hCx37 homotypic gap junction channel does not saturate with transjunctional voltages up to +/-75 mV, nor does it depend linearly on the intracellular electrolyte concentration. The average maximum unitary conductances measured in KCl were 175 pS (30 mM), 236 pS (55 mM), 343 pS (110 mM), and 588 pS (270 mM) in the presence of 0.1 mM MgCl(2). The unexpectedly high unitary conductance at low salt concentrations can be explained by fixed charge groups within or near the channel orifice. Fixed cytoplasmic surface charges (3.4 e) positioned adjacent (15 A) to the channel pore adequately model the data (surface charge density of 0.24 e/(nm)(2)). In other experiments, high Mg(2+) reduced the unitary conductance of hCx37 homotypic gap junction channels more than predicted by screening alone, consistent with specific effects of Mg(2+) on the channel.
The gating behavior of human connexin 37 (hCx37) is unaffected by the nature of the bathing monovalent (for Na, K, Rb). It is modified by [Mg] in the millimolar range. For fitting the kinetics, we propose a simple extension to three states of the canonical 2-state model of the hemichannel. The extra closed state allows for some immobilization of a hemichannel at high transjunctional voltages. The model is reasonably efficient at fitting data at various voltage protocols. Interpreting the fits of the data at different [Mg] is consistent with a binding site for Mg.
A model tissue is proposed in which chemically responsive cells are interconnected by gap junctions and innervated by the autonomic nervous system. The model is explicitly dependent on the following physiologically relevant assumptions: (1) a fraction of the cells are directly innervated, and these cells respond to a periodic neuronal stimulus (i.e. the release of neurotransmitter) by production of an intracellular substance (i.e. second messenger molecule); (2) production of second messenger molecules modulates the amplitude of a cellular response, such as contraction or secretion; (3) intracellular formation of second messenger molecules in innervated cells is proportional to the periodicity of the neuronal stimulus, while the intracellular concentration in non-innervated cells is governed by the half-life of the second messenger molecule and the extent of cell-to-cell coupling; (4) the amplitude of the graded response of the individual cell is related to the intracellular second messenger concentration by a Michaelis-Menten function; (5) the amplitude of the graded tissue response is a function of the innervation density, the frequency of stimulation, and the extent of intercellular coupling. Thus, a stimulus-response relationship was developed, where the magnitude of the tissue response was described as a function of the total tissue stimulus. The predicted stimulus-response curve was encapsulated by two parameters: (1) the Hill-exponent, which reflects the steepness of the stimulus-response curve; and (2) the location of the stimulus-response curve, or the half-maximally effective stimulus. Both random and uniform neuronal innervation patterns were considered in model tissues with various effective dimensions. The simulations were also applied to a realistic model of vascular tissue. The shape of the stimulus-response curve is critically dependent on the geometry of innervation. For physiologically relevant (10-90% over 2-3 orders of magnitude) dose-response curves, the model yields an implicit relationship between three different dimensionless parameters. If, in a system, two of these parameters are known, the model can be used to bracket the possible range of the third parameter.
James Bryan Herrick was an American physician who made several contributions to clinical medicine. Three of his most important observations are described.
The dependence of macroscopic gap junctional conductance (g(j)) upon transjunctional voltage (Vj) was examined in 39 paired osteoblast-like (OB) cells from primary cultures using the double whole cell patch clamp technique. OB cells were derived from calvarial explants of new-born rats. Instantaneous current-voltage (Ij-Vj) relationships of OB cell pairs (n = 6) were linear in the entire voltage range (-150 < Vj < 150 mV) examined. The steady-state Ij-Vj relationship was non-linear for V > or = +/-60 mV. The curve for the normalised steady-state junctional conductance-voltage relationship (Gss/G0-Vj) was bell-shaped, and was fitted with a two-state Boltzmann equation with a minimum conductance (Gmin) of 0.2-0.3, and a half deactivation voltage (Vo) of +/-83 mV. In two recordings unitary gap junction channel activity was observable. The linear I-V relationships revealed a single channel conductance of approximately 100 pS. Application of parathyroid hormone (10(-8) M) had no effect on the voltage dependence nor the magnitude of macroscopic currents (n = 7).
Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage (Gj/Vj) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The Gj/Vj data imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.
Gap junction channels are an integral part of the conduction or propagation of an action potential from cell to cell. Gap junctions have rather unique gating and permeability properties which permit the movement of molecules from cell to cell. These molecules may not be directly linked to action potentials but can alter nonjunctional processes within cells, which in turn can affect conduction velocity. The data described in this review reveal that, for the majority of excitable cells, there are two limiting factors, with respect to gap junctions, that affect the conduction/propagation of action potentials. These are (1) the total number of channels and (2) the selective permeability of the channels. Interestingly, voltage dependence and the time course of voltage inactivation (kinetics) are not rate limiting steps under normal physiological conditions for any of the connexins studied so far. Only specialized rectifying electrical synapses utilize strong voltage dependence and rapid kinetics to permit or deny the continued propagation of an action potential.
The gating parameters of human connexin 43 (Cx43) gap junction channels were determined using dual whole cell patch clamp and methods designed for analysis of multichannel recordings. Under steady-state conditions, the mean open time (MOT) of Cx43 gap junction channels was computed and it ranged from 0.43 to 5.25 s. The computed mean closed times (MCT) varied from 0.21 to 1.49 s. Analysis showed that, while the MOT declined with increasing transjunctional voltage (Vj), the apparent decline in the MCT with Vj was not statistically significant. The calculated open probabilities ranged from 0.50 to 0.95. Inspection of the data showed that there was a prolonged decay in junctional current, which had a time course of 60-150 s. The analysis excluded the possibility of a homogeneous voltage inactivated/deactivated population of independent and identical Cx43 gap junction channels. The analysis provided evidence for a homogeneous population of Cx43 channels, which can mode shift under the influence of voltage. The latter case cannot be distinguished from a heterogeneous population of Cx43 channels in which one population is voltage inactivated/deactivated and another is unaffected or weakly inactivated/deactivated by voltage.
Gap junctions are aqueous intercellular channels formed by a diverse class of membrane-spanning proteins, known as connexins. These aqueous pores provide partial cytoplasmic continuity between cells in most tissues, and are freely permeable to a host of physiologically relevant second messenger molecules/ionic species (e.g., Ca2+, IP3, cAMP, cGMP). Despite the fact that these second messenger molecules/ionic species have been shown to alter junctional patency, there is no clear basis for understanding how dynamic and transient changes in the intracellular concentration of second messenger molecules might modulate the extent of intercellular communication among coupled cells. Thus, we have modified the tissue monolayer model of Ramanan and Brink (1990) to account for both the up-regulatory and down-regulatory effects on junctions by second messenger molecules that diffuse through gap junctions. We have chosen the vascular wall as our morphological correlate because of its anisotropy and large investment of gap junctions. The model allows us to illustrate the putative behavior of gap junctions under a variety of physiologically relevant conditions. The modeling studies demonstrated that transient alterations in intracellular second messenger concentrations are capable of producing 50-125% changes in the number of cells recruited into a functional syncytial unit, after activation of a single cell. Moreover, the model conditions required to demonstrate such physiologically relevant changes in intercellular diffusion among coupled cells are commonly observed in intact tissues and cultured cells.
We present an exact solution to the linearized Nernst-Planck-Poisson equation for spherically symmetric current flow. This solution differs from Levitt's solution (Levitt, D. G. 1992. Biophys. J., Eq. A5) by its dependence on an additional parameter, which is equal to the net ion flux for monovalent ion-selective channels. For ion-selective channels, this solution may provide better boundary conditions to modelling the flow in the channel pore itself, although only at low salt concentrations. We use the solution to estimate the effects of flux interaction between closely packed channels.
Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the open-closed gating of an aqueous pore permeable to ions and molecules of up to 1 kDa or 10-14 A in diameter. We symmetrically altered the ionic composition or asymmetrically added 6-carboxyfluorescein (6-CF, M(r) = 376), a fluorescent tracer, to pairs of connexin37-transfected mouse neuro2A cells to examine the ionic and dye permeability of human connexin37 channels. We demonstrate that the 300-pS channel formed by connexin37 has an effective relative anion/cation permeability ratio of 0.43, directly converts to at least one intermediate (63 pS) subconductance state, and that 6-CF dye transfer is accompanied by a 24% decrease in unitary channel conductance. These observations favor a new interpretation of the gap junction pore consistent with direct ion-channel interactions or electrostatic charge effects common to more conventional multistate ion channels. These results have distinct implications about the different forms of intercellular signaling (cationic, ionic, and/or biochemical) that can occur depending on the expression and conformation of the connexin channel proteins.
Substates which can last up to several seconds are found in the 100-pS channel of the earthworm septum, a putative gap junction channel. The conductance of these substates is highly variable from preparation to preparation, and they are found at almost every fraction of the whole channel conductance. Another phenomenon seen in multichannel recordings is the "conductance shift": here the current passed by several open channels differs from an integral multiple of the current when only one channel is open. These shifts can be modelled by 1) a resistance in series with the channel or 2) long-lived substates. Each of these models fails in particular cases to explain either the magnitude or direction of the shifts. It is possible that both effects are simultaneously present.
We present a proof that the mean open (and closed) times of the individual channels in a multichannel record can be found in a model-independent fashion. As the results are model independent, they can be derived by assuming the simplest model for all the channels, namely that they all have the basic CLOSED in equilibrium with OPEN scheme. In particular, the method can be applied to patches where the channel population is heterogenous with respect to open probability. Multichannel simulations are performed to test the limits of applicability of this method to restricted amounts of data. One conclusion is that increasing the number of channels does not substantially reduce the errors in estimating the mean times, in spite of the 'increased information' present. We also prove the general applicability of the algorithm of Fenwick et al. (1982) in estimating the mean times without knowledge of the number of channels present, and discuss its limitations. An illustration using experimental data is also given.
We have studied multichannel patch-clamp recordings in earthworm axon septal membranes that contain gap junctions. Though all channels have the same conductance and selectivity, the probabilities of the conductance levels in the majority of the recordings could not be fit by assuming independent and identical channels; in these cases, we found that at least two different open probabilities were required to explain the data. The data thus suggest that, within one junctional membrane complex, there exists a heterogenous channel population of similar but not identical channel types. The analysis also revealed cases where cooperativity between individual channels was the only explanation for the amplitude histograms of the observed multichannel activity. The conclusions drawn are based on a theoretical analysis of multichannel current-amplitude histograms. We derive two tests for independent and identical channels. We analyze the effects of mode shifting. These results are based on the ratio of peaks in the histograms; they are independent of the number of channels in the patch and the model of channel gating. In some cases failure to fulfill the criteria of these tests implied an interdependence or cooperativity between channels. Lastly, we have devised statistical tests for stability of the recording in the presence of variance due to finite sample size.
Analytic solutions are found for an infinite chain of cells coupled by gap junctions under two initial conditions: (a) One inner cell initially filled uniformly to a fixed concentration and (b) inner cell maintained indefinitely at constant concentration. The solution can be extended by the product method (Carslaw and Jaeger. 1959. Conduction of Heat in Solids. Oxford University Press.) to monolayers. We can also incorporate leakage through the plasma membrane by the product method. We demonstrate the utility of these results by fitting diffusion data from the septate axon of earthworm and by plots of theoretical profiles from monolayers of cells. Use of these analytic solutions enables one to overcome the limitations of methods that lump the effects of cytoplasmic diffusion and junctional permeability into an effective diffusion coefficient.