Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count

Abstract

A delayed rectifier voltage-gated K(+) channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv alpha and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3(/) mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X(1)- and thromboxaneA(2) receptor-evoked [Ca(2+)](i) increases and delayed the onset of store-operated Ca(2+) influx. Megakaryocyte development was normal in Kv1.3(/) mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca(2+) increases and influences circulating platelet numbers.

Figure 1. Kv1.3 forms the voltage-gated K⁺ channel of human platelets and murine megakaryocytes

A, expression of Kv subunits relative to GAPDH in human platelets. Of all 51 known Kv subunits (see Supplementary information) only three (KCNA3, KCNAB2 and KCNE3) were detected by quantitative PCR at levels above background. B and C, typical whole-cell currents (B) and average peak current densities (C) in response to 3 s duration voltage steps from −80 mV to potentials in the range −120 to 60 mV (see voltage protocol in B) for wild-type (WT) megakaryocytes, Kv1.3-deficient megakaryocytes (Kv1.3^−/−) and WT megakaryocytes after 10 min exposure to 10 nm margatoxin (mgtx).

Figure 2. Kv1.3 is the major determinant of the membrane potential in mouse megakaryocytes

A, current clamp recording of membrane potential in a wild type megakaryocyte during exposure to 10 nm margatoxin (mgtx). B, average membrane potential in wild-type megakaryoctyes in the presence and absence of margatoxin at the times indicated after transition to whole-cell recording, and in Kv1.3^−/− megakaryocytes. C, time-course of block of voltage-gated K⁺ currents by 10 nm margatoxin in 4 different wild-type megakaryocytes.

Figure 3. Role for Kv1.3 in human platelet Ca²⁺ signalling

A and B, typical (a) and average (b) [Ca²⁺]_i responses in fura-2-loaded washed human platelet suspensions in the presence and absence of 10 nm margatoxin (mgtx). A. Responses following activation of P2X₁ and thromboxane A₂ receptors by αβmeATP (1 μm) and U46619 (500 nm), respectively. B, store-operated [Ca²⁺]_i increases induced by addition of CaCl₂ (0.25 mm) after 10 min in 1 μm thapsigargin; [Ca²⁺]_i increases were measured after 10 s and at the peak of the response.

Figure 4. Role for Kv1.3 in platelet count, but not megakaryocyte development

A, megakaryocyte size distribution in marrow from wild-type and Kv1.3-deficient mice; Aa, sample sections stained with a FITC-conjugated anti-CD41 antibody, and Ab, frequency histogram of different size megakaryocytes, which showed no significant differences (P > 0.05) across the range of sizes. B, specific membrane capacitance measurement of the demarcation membrane system in Kv1.3^−/− and wild-type megakaryocytes (n= 23 and 61, respectively; P > 0.05). C, platelet count in wild-type and Kv1.3-deficient mice (n= 7).