Puljung MC , Berthoud VM , Beyer EC , Hanck DA .

DA-07255 NIDA NIH HHS , F31NS-42972 NINDS NIH HHS , HL-45466 NHLBI NIH HHS , R01 HL059199-05A2 NHLBI NIH HHS , R01 HL059199 NHLBI NIH HHS , T32 DA007255 NIDA NIH HHS , R01 HL045466 NHLBI NIH HHS , F31 NS042972 NINDS NIH HHS

	Figure 1. . Nonjunctional currents from hCx37 expressed in Xenopus laevis oocytes. (A, top) Currents resulting from a series of voltage steps from a holding potential of −40 mV in 10-mV increments to a potential of +90 mV in an oocyte injected with RNA encoding hCx37 plus AntiCx38. (A, bottom) Currents from an oocyte injected only with AntiCx38. Currents were recorded in 0DivOR2 plus 1 mM Ca2+. (B) Average, isochronal (see materials and methods) current–voltage relationship for oocytes injected with hCx37 RNA plus AntiCx38 (▪, n = 10) and oocytes injected only with AntiCx38 (○, n = 3). Currents were recorded in 0DivOR2 plus 0.5 mM Ca2+. (C) Normalized, average, isochronal current–voltage relationships for oocytes injected with hCx37 RNA plus AntiCx38, recorded in 0DivOR2 plus 0.5 mM Ca2+ in the presence (•, n = 3) or absence (▪, n = 10) of 10 mM 1-heptanol. Currents in the absence of heptanol were corrected by subtracting the mean of the oocytes injected only with AntiCx38. Currents in the presence of heptanol were not corrected by the control currents, since the endogenous sodium current in oocytes was completely inhibited by 10 mM 1-heptanol (not depicted). All currents were normalized to the peak current at +90 mV in the absence of 1-heptanol. The current–voltage relationships were all leak corrected.
	Figure 2. . hCx37 hemichannel currents lose their voltage and time-dependent properties in oocytes following wash-out of divalent cations. (A) Representative currents resulting from a series of steps from a holding potential of −40 mV to potentials ranging from −100 to +90 mV in 10-mV increments for an oocyte injected with hCx37 RNA along with AntiCx38 (left) and an oocyte injected with AntiCx38 alone (right) stored in 0DivOR2. (B) Average, isochronal current–voltage relationships over the voltage range from −100 to +90 mV for oocytes injected with hCx37 RNA plus AntiCx38 (▪, n = 6) and oocytes injected only with AntiCx38 (○, n = 5). (C) Average, isochronal current–voltage relationships over the voltage range from −100 to +10 mV for oocytes injected with hCx37 RNA plus AntiCx38 (▪, n = 30) and oocytes injected only with AntiCx38 (○, n = 20). (D) Bar graph showing macroscopic hCx37 hemichannel conductance (assessed between −60 and 0 mV) as a function of the amount of hCx37 RNA injected into the oocyte (n = 2, 60 ng; n = 3, 12 ng; n = 4, 6 ng; n = 4, 3 ng; n = 4, 0.3 ng). The asterisk corresponds to current levels that were significantly larger than those with 0.3 ng injected (P < 0.0001, Student's t test).
	Figure 3. . hCx37 hemichannel currents are reversibly inhibited by divalent cations. Representative currents resulting from a series of voltage steps from a holding potential of −40 mV to potentials ranging from −100 to +10 mV in 10-mV increments for oocytes injected with hCx37 RNA along with AntiCx38 (A and C) and an oocyte injected only with AntiCx38 (B). (A) Currents from hCx37 hemichannels before (left), during treatment with (middle), and after wash-out of (right) 1 mM Ca2+. (B) Currents from an oocyte injected only with AntiCx38 before (left), during treatment with (middle), and after wash-out of (right) 1 mM Ca2+. (C) Currents from hCx37 hemichannels before (left), during treatment with (middle), and after wash-out of (right) 20 mM Mg2+. (D) Average, isochronal current–voltage relationships for oocytes injected with hCx37 RNA plus AntiCx38 and stored in 0DivOR2. Recordings were made before (▪, n = 8), during treatment with (•, n = 8), and after wash-out of (▴, n = 8) 1 mM Ca2+. (E) Average, isochronal current–voltage relationships for oocytes injected with AntiCx38 alone and stored in 0DivOR2. Recordings were made before (▪, n = 3), during treatment with (•, n = 3), and after wash-out of (▴, n = 3) 1 mM Ca2+. (F) Average, isochronal current–voltage relationships for oocytes injected with hCx37 RNA plus AntiCx38 and stored in 0DivOR2. Recordings were made before (▪, n = 6), during treatment with (•, n = 6), and after wash-out of (▴, n = 6) 20 mM Mg2+. (G) Concentration–response curves (see materials and methods) showing inhibition of hCx37 hemichannel current at −40 mV as a function of [Ca2+] (•) and [Mg2+] (▪). The IC50 for Ca2+ was 107 μM with a Hill coefficient of 3.1 ± 0.5 (20 μM, n = 3; 100 μM, n = 3; 200 μM, n = 5; 1 mM, n = 4; 2 mM, n = 6). The IC50 for Mg2+ was 1.30 mM with a Hill coefficient of 4.5 ± 1.1 (500 μM, n = 5; 1 mM, n = 6; 2 mM, n = 4; 5 mM, n = 3; 10 mM, n = 5; 20 mM, n = 4).
	Figure 4. . The rate of divalent cation inhibition of hCx37 hemichannel currents was concentration dependent. (A) Representative trace showing current from an individual oocyte injected with hCx37 RNA plus AntiCx38. The disappearance of current (left) is upon wash-in of 1 mM Ca2+. The reappearance of current (right) is upon wash-out of Ca2+. These recordings were performed at a holding potential of −40 mV. Both wash-in and wash-out traces were fit with single exponential curves to determine kmod. The time courses were fit with an equation of the form I = I∝ − Ae−kt, where I is the current, I∝ is the amount of current at steady state, A is the amplitude, t is time, and k is kmod (see results). The concentration dependence of kmod for hCx37 hemichannel current at −40 mV is shown for Ca2+ (B) and Mg2+ (C). Both plots were fit with straight lines. For Ca2+, r = 0.86; 0 mM, n = 4; 0.1 mM, n = 2; 0.2 mM, n = 5; 1 mM, n = 5; 2 mM, n = 3. For Mg2+, r = 0.82; 0 mM, n = 2; 1 mM, n = 4; 2 mM, n = 1; 5 mM, n = 4; 10 mM, n = 5, 20 mM, n = 2.
	Figure 5. . Gd3+ inhibits hCx37 hemichannel current. (A) Representative currents resulting from a series of steps from a holding potential of −40 mV to potentials ranging from −100 to +10 mV in 10-mV increments for an oocyte injected with hCx37 RNA along with AntiCx38. The currents were recorded before (left) and after (right) the addition of 100 μM Gd3+ to the bath. (B) Bar graph showing kmod for hCx37 hemichannel current inhibition by two different concentrations of Gd3+, 100 μM (n = 4) and 200 μM (n = 4).
	Figure 6. . Polyvalent cation inhibition of hCx37 hemichannels is voltage dependent. (A) Plot of kmod for hCx37 hemichannel current inhibition by 200 μM Ca2+ as a function of potential (▪; −110 mV, n = 1; −100 mV, n = 4; −90 mV, n = 3; −80 mV, n = 4; −60 mV, n = 5; −40 mV, n = 4; −20 mV, n = 4). The curve is a single exponential decay fit to the data of the form kmod = kpos + Ae−V/dV, where kpos is the minimum kmod at more positive potentials, A is the amplitude, V is voltage, an dV is the amount in millivolts necessary to change kmod by a factor of e. The open circles (○) represent koff as a function of potential (−100 mV, n = 2; −80 mV, n = 3; −60 mV, n = 6; −40 mV, n = 5; −20 mV, n = 3). The straight line shows the mean koff of 0.0035 s−1. The inset shows a plot of the percent inhibition by 200 μM Ca2+ as a function of holding potential (−100 mV, n = 4; −60 mV, n = 4; −40 mV, n = 5; −20 mV, n = 3). The curve in the inset represents the predicted percentage block as a function of potential, based on a model of voltage-dependent block for which steady-state block (ssblock) is given by the equation ssblock = 100/(1 + 10((log(IC50) − log[Ca])*p)), where IC50 is predicted by the fits to the measured kon and koff as a function of potential (A, C, and D) and p is the Hill coefficient for Ca2+ binding (∼3.1). Data from the inset were leak corrected by the mean of the AntiCx38-injected oocytes from the day each data point was acquired (n = 2–4). The data were normalized to block at −100 mV where close to 100% block is predicted by the above equation for 200 μM Ca2+. (B) Plot of kmod for hCx37 hemichannel current inhibition by 1 mM Mg2+ as a function of potential (▪; −100 mV, n = 2; −90 mV, n = 1; −80 mV, n = 2; −70 mV, n = 1; −60 mV, n = 4; −50 mV, n = 2; −40 mV, n = 9). The curve is a single exponential decay fit to the data of the form shown in A. The open circles (○) represent koff as a function of potential (−60 mV, n = 4; −40 mV, n = 2). The straight line shows the mean koff of 0.0024 s−1. (C) Plot of kmod for hCx37 hemichannel current inhibition by 100 μM Gd3+ as a function of potential (−110 mV, n = 2; −100 mV, n = 7; −90 mV, n = 3; −80 mV, n = 5; −60 mV, n = 3; −40 mV, n = 4). The curve is a single exponential decay fit to the data of the form shown in A. (D) Plot of kon for Ca2+ (▪), Mg2+ (•), and Gd3+ (▴) ions as a function of holding potential. The curves are single exponential decays fit to the data of the form kon = kpos + Ae−V/dV (see results).
	Figure 7. . Activation rate of hemichannel current depends on the ions in the bathing solution. (A) Currents resulting from a series of voltage steps from a holding potential of −40 mV in 10-mV increments to a potential of +90 mV applied to oocytes injected with hCx37 RNA plus AntiCx38, recorded in 0DivOR2 plus 1 mM Ca2+ (left) or 200 μM Gd3+ (right). These currents were fit with the sum of two exponentials to determine rate constants (see results). (B) Fast activation rate constant (kfast) as a function of potential for hCx37-expressing oocytes recorded in 0DivOR2 containing 1 mM Ca2+ (○; +40 mV, n = 8; +50 mV, n = 8; +60 mV, n = 8; +70 mV, n = 9; +80 mV, n = 8; +90 mV, n = 8; +100 mV, n = 8) or 200 μM Gd3+ (▪; +60 mV, n = 15; +70 mV, n = 16; +80 mV, n = 16; +90 mV, n = 16; +100 mV, n = 10). (C) Slow activation rate constant (kslow) as a function of potential for hCx37-expressing oocytes recorded in 0DivOR2 containing 1 mM Ca2+ (○; +40 mV, n = 3; +50 mV, n = 7; +60 mV, n = 6; +70 mV, n = 7; +80 mV, n = 7; +90 mV, n = 8; +100 mV, n = 8) or 200 μM Gd3+ (▪; +60 mV, n = 15; +70 mV, n = 16; +80 mV, n = 16; +90 mV, n = 16; +100 mV, n = 10). (D) kfast (upper) and kslow (lower) for hCx37 hemichannel current recorded in 0DivOR2 containing 1 mM Ca2+ (○) or 0.5 mM Ca2+ (▴). For kfast in 0.5 mM Ca2+, at +40 mV, n = 10; +50 mV, n = 9; +60 mV, n = 10; +70 mV, n = 10; +80 mV, n = 10; +90 mV, n = 9; and +100 mV, n = 10. For kslow in 0.5 mM Ca2+, at +40 mV, n = 1; +50 mV, n = 6; +60 mV, n = 10; +70 mV, n = 10; +80 mV, n = 10; +90 mV, n = 10; and +100 mV, n = 10. (E) kfast (upper) and kslow (lower) for hCx37 hemichannel current recorded in 0DivOR2 containing 200 μM Gd3+ (▪) or 100 μM Gd3+ (○). For kfast in 100 μM Gd3+ at +60 mV, n = 5; +70 mV, n = 6; +80 mV, n = 6; +90 mV, n = 6; and +100 mV, n = 6. For kslow in 100 μM Gd3+ at +60 mV, n = 2; +70 mV, n = 6; +80 mV, n = 6; +90 mV, n = 6; and +100 mV, n = 5.
	Figure 8. . Current deactivation depends on polyvalent concentration and flux during channel opening. (A) Fast and slow rate constants of deactivation in 0.5 mM Ca2+ as a function of the length of the depolarizing pulse to +90 mV. At 10 s, n = 10, at 20 s, n = 9, and at 40 s, n = 6. The inset shows representative current traces that are the response to depolarization for 5, 10, 20, and 40 s to +90 mV from a holding potential of −40 mV, followed by repolarization to −40 mV. (B) Fast and slow rate constants of deactivation at −40 mV following 40-s depolarizing pulses to +90 mV as a function of extracellular Ca2+ concentration. The inset shows representative tail currents recorded upon repolarization to −40 mV in 0DivOR2 plus 1.0 mM Ca2+ and 0.5 mM Ca2+. For 0.5 mM Ca2+, n = 6; for 1.0 mM Ca2+, n = 9. (C) Fast (triangles) and slow (circles) rate constants for deactivation at −40 mV as a function of the amount of charge passed during 40-s depolarizing pulses to +90 mV (filled symbols) or +70 mV (open symbols). Each data point represents an individual experiment. A total of six oocytes was used to gather the data. The data were fit to straight lines (kfast, r = 0.9; kslow, r = 0.7). The inset shows representative current traces corresponding to depolarization from a holding potential of −40 to +70 mV and +90 mV followed by repolarization to −40 mV in a single oocyte. Data were acquired at a sampling rate of 100 Hz and filtered at 50 Hz. Rate constants were corrected by the koff for Ca2+ at negative potentials (0.0035 s−1) to yield a value of kon*[Ca2+].
	Figure 9. . Gating model for hCx37 hemichannels. Illustration of the gating process for hCx37 hemichannels. At negative potentials (left) the channel is occupied with several polyvalent cations. These bind to a site on the cytoplasmic side of the channel, blocking current flow. At positive potentials (center) the koff for the polyvalent cations becomes significant, the ions are ejected from the site of block, and current is allowed to flow. Polyvalent cations remain at a high local concentration. Upon extended depolarization (right), the local concentration is depleted as well. Upon repolarization, the polyvalent cations bind the channel again, inhibiting current flow (left). A kinetic model for hCx37 hemichannel gating is shown below. Cx is the blocked hemichannel, Cx* is the unblocked hemichannel, Cx** is the unblocked hemichannel free of any polyvalent cations, M+ is a polyvalent cation, n is the minimum number of polyvalent cations needed to inhibit current (see discussion).
	Figure 10. . Polyvalent cation effects are not due to surface charge screening. (A) Simulated conductance–voltage curves. Each curve is shifted 40 mV relative to the next. Increasing numbers correspond to increasing amounts of polyvalent cations (see discussion). The inset shows current–voltage relationships corresponding to the conductance–voltage relationships. The bold lines in both the figure and inset correspond to the maximal conductance over the voltage range depicted. (B) Plot of the conductance–voltage relationship for hCx37 in 0DivOR2 plus 0.02 mM Ca2+ (▪, n = 6), 0.1 mM Ca2+ (•, n = 4), 0.2 mM Ca2+ (▴, n = 6), and 1 mM Ca2+ (▾, n = 4). The corresponding, isochronal current–voltage relationships, shown in the inset, represent current responses to the voltage protocol shown in Fig. 2. Conductance was determined at each potential from the slope of the current–voltage relationship between adjacent points. The dashed lines in the inset are predicted currents after 2.5-s voltage pulses based on the expectation from steady-state block in Fig. 6. In brief, the steady-state block was determined for −40 mV, and each new test potential. These values were connected by single exponentials with kmod determined by the measured koff and kon (Fig. 6) and the [Ca2+]. The predicted amount of block at 2.5 s was multiplied by an open-channel, current–voltage relationship predicted by a line formed between the current at −40 mV, divided by the fractional block at that potential (Fig. 3 G) and the origin.

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