Srinivas M et al. (2006), Regulation of connexin hemichannels by monovale...

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J Gen Physiol 2006 Jan 01;1271:67-75. doi: 10.1085/jgp.200509397.

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Regulation of connexin hemichannels by monovalent cations.

Srinivas M , Calderon DP , Kronengold J , Verselis VK .

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Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.

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Species referenced: Xenopus laevis
Genes referenced: gja3 gja8

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	Figure 1. Membrane currents in Xenopus oocytes expressing Cx50 (middle) and Cx46 (bottom) respond differently to changes in external calcium. MND96 solutions with external Ca2+ concentrations of 1.8, 0.6, and 0.2 mM were applied to oocytes voltage clamped at −40 mV (Vhold). A pair of 5-s voltage steps, one depolarizing to +50 mV and one hyperpolarizing to −110 mV were applied at each Ca2+ concentration. Reducing external Ca2+ led to the development of a progressively larger inward current at Vhold in Cx50-expressing oocytes that was accompanied by progressively larger currents in response to both polarities of applied voltage steps. In contrast, little or no change in current at Vhold or in response to hyperpolarizing voltage steps were observed in Cx46-expressing oocytes upon reducing external Ca2+. However, currents elicited by the depolarizing steps were progressively larger as Ca2+ was reduced, consistent with the effects of Ca2+ previously described (Ebihara and Steiner, 1993).
	Figure 2. External monovalent cations strongly potentiate Cx50, but not Cx46, hemichannel currents. (A) Membrane currents were monitored in a Cx50-expressing oocyte voltage clamped at −40 mV. Reduction of Ca2+ from 1.8 mM to 0.2 mM in a Na+ solution promoted opening of Cx50 hemichannels evident by the development of an inward current. Equimolar replacement of Na+ with K+ or Cs+ (gray bars) in the continued presence of 0.2 mM Ca2+ caused a large increase in the amplitude of the inward current at −40 mV, an effect that was reversible upon switching back to Na+. (B) Bar graph summarizing the effect of replacing external Na+ with a variety of monovalent cations on Cx50 hemichannel currents. K+, Rb+, NH4+, and Cs+, but not TEA+, Li+, and choline, caused a robust potentiation in current. Bars represent the means ± SEM of the fold change caused by each monovalent cation (n ranged from 4 to 15). Mean values in K+ and Cs+ were significantly different (P < 0.05). (C) Cx46 hemichannels were only moderately affected by replacing external Na+ with K+. Bar graph shows a comparison of the increase in Cx46 (white bars) and Cx50 (solid bars) hemichannel currents upon replacing external Na+ with K+. Because Cx46 activates only at positive voltages, current amplitudes were measured at the ends of 5-s depolarizing voltage steps to +50 mV. Such changes were not observed in uninjected oocytes (gray bars). Each bar represents the mean ± SEM of six oocytes.
	Figure 3. Unitary conductance and gating properties of Cx50 hemichannels do not differ in NaCl or KCl solutions in the absence of Ca2+. (A and B) Single Cx50 hemichannel currents obtained with 8-s voltage ramps from −70 to 70 mV applied to excised patches in symmetric KCl (A) or NaCl (B) solutions each containing 1 mM Ca2+/5 mM EGTA to maintain the free Ca2+ concentration <10−7 M. (C) Single Cx50 hemichannel currents obtained with 8-s voltage ramps from −65 to 65 mV applied to excised inside-out patches in symmetric NaCl solutions (left) or asymmetric KCl(in)/NaCl(out) solutions (center). Recordings are from the same patch. Segments of the I-V relationships are expanded to show the small shift in Erev −2.6 mV (right). (D and E) Conductance–voltage (G-V) relationships of Cx50 hemichannels (shown on right in D and E) constructed from ensemble (n = 20) averaged currents (shown on left in D and E) in response to ±70 mV, 8-s voltage ramps applied to cell attached patches. Pipette solutions containing KCl (D) or NaCl (E) were the same as in A and B.
	Figure 4. Potentiation by K+ is dependent on external Ca2+. (A) Example of recordings of membrane currents in a Cx50-expressing oocyte voltage clamped at −40 mV over a range of Ca2+ concentrations in NaCl and KCl. For each series, the oocyte was placed in 100 mM NaCl containing 2 mM Ca2+ and sequentially exposed to 100 mM NaCl (top panel, dashes) or 100 mM KCl (bottom panel, dashes) containing Ca2+ concentrations of (in mM) 1.0, 0.6, 0.25, 0.1, and 0.05 (open bars). Between each exposure, oocytes were returned to 100 NaCl containing 2 mM Ca2+ (filled bars). Saturation of the effect of Ca2+ was achieved at a higher external Ca2+ concentration in K+. (B) Bar graph showing the magnitude of potentiation of Cx50 hemichannel currents upon replacement of external Na+ with K+ at different Ca2+ concentrations. The effect of external K+ was markedly reduced at the lower end of the Ca2+ concentration range examined. Current magnitudes due to the differences in mobility between Na+ and K+ are not corrected. Each bar represents the mean ± SEM of 6–15 oocytes. (C) Concentration response curves for Ca2+ inhibition of Cx50 hemichannel currents in external Na+ (squares) and K+ (inverted triangles) solutions. For Na+ and K+, current amplitudes at each Ca2+ concentration were normalized to the responses obtained at 0.05 mM Ca2+ in Na+ and K+, respectively. Solid lines are fits of the data to the Hill equation of the form Normalized I = Kdn/(Kdn + [Ca2+]n. Kd and n values were 259 μM and 2.2 in Na+ and 580 μM and 3.2 in K+. (D) Bar graph showing that the inhibition of Cx50 hemichannel currents by other divalent cations, Ni2+ and Co2+, is also affected by replacement of Na+ with K+. Each bar represents the mean ± SEM of three to four oocytes.
	Figure 5. The difference in the sensitivity of Cx46 and Cx50 hemichannels to external K+ resides in the NH2-terminal half. (A) Shown are hemichannel currents upon replacing external Na+ with K+ in oocytes expressing Cx4650NT-CL and Cx5046NT-CL chimeric hemichannels. Oocytes were voltage clamped at −40 mV. Currents in oocytes expressing Cx4650NT-CL, in which Cx46 sequence from the NT through the CL was replaced with Cx50, were strongly potentiated, much like Cx50, when Na+ was replaced with K+. The reciprocal chimera, Cx5046NT-CL, was similar to Cx46 in being largely insensitive to external K+, except for a small shift in the baseline current that was also observed in uninjected oocytes. (B) Hemichannel currents in oocytes expressing Cx5046NT-CL in response to hyperpolarizing and depolarizing pulses to −110 and +50 mV, respectively, applied from holding potential of −40 mV in Na+- and K+-containing external solutions. Membrane currents in Cx5046NT-CL–expressing oocytes qualitative resemble those in Cx46-expressing oocytes and show an increase in external K+ similar to that of Cx46 hemichannels.

References [+] :

Adelman, Blocking of the squid axon potassium channel by external caesium ions. 1978, Pubmed