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Biochem Biophys Res Commun
2022 Jan 01;586:107-113. doi: 10.1016/j.bbrc.2021.11.052.
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Direct inhibition of CaV2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction.
Contreras GF
,
Saavedra J
,
Navarro-Quezada N
,
Mellado G
,
Gonzalez C
,
Neely A
.
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The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.
Fig. 1. Gem but not Rem inhibit CaV2.3 channels currents. A) Macroscopic current recorded from oocytes injected 5 days earlier with the following cRNAs combinations: CaV2.3 (blue) alone or with Gem (orange) or Rem (purple) during the stimulation protocol showed as inset. B) average maximal inward currents for CaV2.3 alone was −0.53 ± 0.38 μA (n = 4), for CaV2.3 +Gem −0.01 ± 0.00 μA (n = 4) and −0.51 ± 0.18 μA (n = 6) for or CaV2.3 +Rem.C) Representative macroscopic currents from oocytes injected with CaV2.3 cRNA (blue), recorded 30 min after protein injection of Gem or Rem. Each trace was obtained during a 150-ms pulse of increasing amplitude, starting at −90 mV and ending at 70 mV in 10-mV increments. Membrane potential was held at −90 mV until the beginning of the pulse and returned to −40 mV for the remaining of the trace. D) Current density versus voltage relationship from oocytes expressing CaV2.3 alone (Blue) or together with either Gem (orange) or Rem (purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Gem as recombinant protein inhibits CaV2.3 currents within a short time A) Time dependence of current inhibition recorded every 5 min after injection of recombinant Gem (orange, n = 4) or buffer (blue, n = 6). Data shown as circles correspond to the average of maximal inward current obtained from the IV normalized to each recording's first measurement. The shaded area covers the mean + −SEM). The blue and orange dash line is a linear regression with intercept constrained to 1. The slopes were −1.8 ± 0.01%/min and −4.7 ± 0.2%/min for buffer and Gem injected oocytes, respectively. B) IV curves from oocytes recorded immediately, 10, and 20 min after infection of either elution buffer (blue) or Gem orange). C) Representative current traces evoked by 30 ms pulse to −30,0, 30 from oocytes recorded 5- and 90-min following injection of either elution buffer (blue) or Gem (orange). D) Maximal inward current 5 or 90 after injection of either elution buffer (blue) and Gem (orange) shown as box plots with individual points as circles and estimated means of −0.26 ± 0.02 μA (n = 12) and −0.24 ± 0.02 μA (n = 14) 5 min after injection of buffer or Gem, respectively and, at 90 min following the injection the mean values were −0.21 nA ± 0.05 μA (n = 5) and 0 nA (n = 14) for buffer and Gem, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Gem induce reduction on Qon A) Representative traces of gating current (QON) measured at reversal potential elicited by voltage pulses from 20 to 30 mV in steps of 2 mV. The highlighted trace is the one selected to measure the QON and the tail current. B) Box plots of QON at 5 and 90 min after either buffer or Gem injection with individual points as circles. The estimated means at 5 min were 241 ± 34 pC (n = 12) and 139 ± 12 pC (n = 14) for buffer and Gem, respectively. 90 min after injection, the estimated means were 204 ± 63 pC (n = 7) and 0 pC (n = 14) for buffer and Gem, respectively. C) ITAIL at 5 and 90 min after injection of either buffer or Gem from the same recordings as in (B). Estimated means at 5 min were −1.99 ± 0.16 μA and −1.69 ± 0.11 μA for buffer and Gem, respectively and −2.01 ± 0.41 μA and 0 (n = 19) at 90 min.
Fig. 4. Gem-induced reduction of charge movement prevented by co-expression of dominant-negative dynamin mutant K44A A) Representative traces from oocytes co-expressing CaV2.3 with DynK44A buffer (purple) or Gem (green) injected incubated for 5 and 90 min. Traces evoked by the protocol described in Fig. 1A are superimposed currents. B) Maximal inward currents of oocytes at either 5 or 90 min after injecting Gem or Buffer are shown as box plots with individual points as circles. Average maximal inward currents were −0.11 ± 0.02 μA (n = 5) and −0.13 ± 0.3 μA (n = 7) at 5 min after injection of buffer and Gem, respectively, and at 90 min, currents were reduced to −0.07 ± 0.01 μA (n = 18) and −0.09 ± 0.1 nA (n = 8) with buffer and Gem, respectively. C) Representative traces of gating currents measured at the reversal potential as in Fig. 3C. D) QON Box plots at 5 and 90 min after buffer or Gem injections with estimated means of: 106 ± 9 pC (n = 3) and 110 ± 23 pC (n = 5) at 5 min and 93 ± 34 nC (n = 5) and 79 ± 19 pC (n = 15) at 90 min for buffer or Gem injected, respectively. E) Box plots of ITAIL from the same recordings set as in (D). For buffer injected oocytes, means for ITAIL were −1.1 ± 0.2 μA at 5 min and −0.73 ± 0.1 μA at 90 min. For Gem injected oocytes, at 5 min, ITAIL means was −0.85 ± 0.14 μA compared to −0.85 ± 0.1 μA obtained at 90 min. F) Scatter plot of QON versus ITAIL for oocytes injected with DynK44A injected with Gem (n = 19; green) or buffer (n = 17; purple). The solid lines correspond to the linear regression with intercept fixed at 0. The slope for DynK44A + Gem was −81.34 ± 10 pC/μA and −134.27 ± 20.58 pC/μA for DynK44A + buffer. For comparison, a blue line with a slope of −94.8 pC/μA is included that corresponds to the linear regression between QON and ITAIL obtained from oocytes expressing CaV2.3 alone and injected with buffer. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
